Methods for producing biomass rich in dha, palmitic acid and protein using a eukaryotic microorganism

ABSTRACT

Provided herein are eukaryotic microorganisms having a simple lipid profile comprising long chain fatty acids (LCFAs). Also provided are compositions and cultures comprising the eukaryotic microorganisms as well as methods of using the eukaryotic microorganisms.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/845,697 filed Dec. 18, 2017, which claims priority to U.S.Provisional Application No. 62/437,886, filed Dec. 22, 2016, which isincorporated by reference herein in its entirety.

REFERENCE TO THE SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 15, 2022, isnamed 095523_1283066_019US2_SL and is 32,933 bytes in size.

BACKGROUND

Fish constitute a large portion of dietary protein and provide essentialomega-3 lipids in human diets. Increasing demand is driving growth inseafood markets at an annual rate of 6%. Aquaculture is an integral partof this market as wild catch can no longer satisfy consumer demand.Historically, fish meal (protein) and fish oil (fatty acids, and omega-3fatty acids in particular) have been used extensively. The growth ofaquaculture requires that new, sustainable sources of aquaculture feed,providing carbohydrate, protein and omega-3 fatty acids, be developed.Due to technology limitations, microalgae have been used as amicro-ingredient employed to improve specific properties, not as a macro(protein, oil or carbohydrate) nutrient. Thus, although known to besuitable as a feed component, the use of microalgae is currently limitedas a means for addressing sustainability.

BRIEF SUMMARY

Provided herein are eukaryotic microorganisms having a simple lipidprofile comprising long chain fatty acids (LCFAs). Also provided arecompositions and cultures comprising the eukaryotic microorganisms andheterotrophic medium. Methods of making a lipid composition using thedisclosed eukaryotic microorganisms and methods of using lipidcompositions by incorporating the lipid compositions into foodstuffs arealso provided herein. Also provided are methods of making protein richbiomass using the disclosed microorganisms and optionally incorporatingthe protein rich biomass into foodstuffs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing biomass and TFA production by strain G3-1 inliquid media: basal (B) and full fermentation media (FF).

FIG. 2 is a graph showing the effect of media composition on fatty acidprofile of the intracellular oil of strain G3-1: basal (B), fullfermentation media (FF).

FIG. 3 is a graph showing the effect of hot-TCA treatments on theefficiency of protein extraction (protein content as %) in lyophilizedG3-1 biomass from early exponential (T16h and T22h) and stationary phase(T139h and T189h).

FIG. 4 is a graph showing the fatty acid profile of the intracellularoil of strain G3-1 under different liquid media compositions.

FIG. 5 is a graph showing the fatty acid profile of the intracellularoil of strain G3-1. Cells were cultured in 2-L fermenters using aselected basal media.

FIG. 6 is a graph showing the fatty acid profile of the intracellularoil of strain G3-1. Cells were cultured in 2-L fermenters using amodified full fermentation media.

FIG. 7 is a graph showing the fatty acid profile of the intracellularoil of strain G3-1. Cells were cultured in 2-L fermenters using halfconcentration of ammonium sulphate in a modified full fermentationmedia.

FIG. 8 is a graph showing the fatty acid profile of the intracellularoil of strain G3-1 under different liquid media compositions.

FIG. 9 is a graph showing the fatty acid profile of the intracellularoil of strain G3-1. Cells were cultured in 30 L fermenter using VU1media.

FIG. 10 is a graph showing the fatty acid profile of the intracellularoil of strain G3-1. Cells were cultured in 30 L fermenter using VU2media.

FIG. 11 is a graph showing the fatty acid profile of the intracellularoil of strain G3-1. Cells were cultured in 2 L fermenter using VU3 mediaand crude glycerol as the carbon source.

DETAILED DESCRIPTION

Microalgae are the primary producers of aquatic ecosystems and representthe origin of essential nutrients (e.g., protein and omega-3 fattyacids), which are metabolized and/or bio-accumulated in the aquatic foodchain. As such, high protein and high omega-3 long chain polyunsaturatedfatty acid (LC-PUFA) products derived from microalgae have enormouspotential to sustainably meet the dietary demands of the rapidly-growingaquaculture sector. Among the large variety of microalgae, the use ofheterotrophic microalgae has the greatest potential to provideaquaculture feed inputs that are free from the supply and demandconstraints of plant and animal products. Heterotrophic microalgaeproduction requires significantly less land and water, has betterprocess economics, and is independent of environmental conditions (i.e.,climate independent). For example, volumetric biomass productivity ofheterotrophic microalgae can be two orders of magnitude higher than thatof photosynthetic microalgae. The use of heterotrophic microalgae alsoprovides the opportunity to leverage inexpensive and abundant non-foodcarbon sources converting them directly into high value products throughfermentation. Such production processes can also be more easily scaledup, as would be required for an aquaculture feed product.

Provided herein are eukaryotic microorganisms having a simple lipidprofile comprising long chain fatty acids (LCFAs). Microorganisms,including Thraustochytrids, produce a variety of lipids including fattyacids in various forms and amounts. As used herein, the term lipidincludes phospholipids, free fatty acids, esters of fatty acids,triacylglycerols, sterols and sterol esters, carotenoids, xanthophylls(e.g., oxycarotenoids), hydrocarbons, and other lipids known to one ofordinary skill in the art. Fatty acids are hydrocarbon chains thatterminate in a carboxyl group, being termed unsaturated if they containat least one carbon-carbon double bond, and polyunsaturated when theycontain multiple carbon-carbon double bonds. For example, microorganismscan produce (i) short-chain fatty acids (SCFA), which are fatty acidswith aliphatic tails of fewer than six carbons (e.g., butyric acid);(ii) medium-chain fatty acids (MCFA), which are fatty acids withaliphatic tails of 6-12 carbons; (iii) long-chain fatty acids (LCFA),which are fatty acids with aliphatic tails 13 to 21 carbons; and verylong chain fatty acids (VLCFA), which are fatty acids with aliphatictails longer than 22 carbons. Various microorganisms produce varyingtypes and amounts of these fatty acids. The specific types and amountsof fatty acids are collectively referred to herein in as themicroorganism's lipid profile. Thus, as used herein, the term “lipidprofile” refers to the types of lipids and amounts of lipids produced ina microorganism.

As used herein, a “simple lipid profile” refers to a microorganismhaving 95% or more of the triglycerides in the microorganism being madeup of 1, 2, 3, or 4 of the major long chain fatty acids. Optionally,95%, 96%, 97%, 98%, 99%, or 100% of the triglycerides in themicroorganism comprise 1, 2, 3, or 4 of the major long chain fattyacids. Optionally, 95%, 96%, 97%, 98%, 99%, or 100% of the triglyceridesin the microorganism comprises myristate (C14:0), palmitic acid (C16:0),docosapentaenoic acid n-6 (C22:5n-6, DPAn6), and docosahexaenoic acid(C22:6n-3, DHA). As used herein, a triglyceride refers to a moleculecomposed of three fatty acids covalently linked to a glyceride molecule.Thus, the triglyceride fraction of the total fatty acids in themicroorganism can be comprised of 95%, 96%, 97%, 98%, 99%, or 100%myristate (C14:0), palmitic acid (C16:0), docosapentaenoic acid n-6(C22:5n-6, DPAn6), and docosahexaenoic acid (C22:6n-3, DHA).

Long chain fatty acids (LCFA) include, but are not limited to, myristate(C14:0), palmitic acid (C16:0), docosapentaenoic acid n-6 (C22:5n-6,DPAn6), docosahexaenoic acid (C22:6n-3, DHA), lauric acid (C12:0),pentadecylic acid (C15:0), palmitoleic acid (C16:1), margaric acid(C17:0), stearic acid (C18:0), vaccenic acid (C18:1n-7), oleic acid(C18:1n-9), γ-linolenic acid (C18:3n-6), α-linolenic acid (C18:3n-3),stearidonic acid (C18:4), arachidic acid (C20:0), dihomo-γ-linolenicacid (C20:3n-6), arachidonic acid (C20:4n-6, ARA), eicosapentaenoic acid(C20:5n-3, EPA), behenic acid (C22:0), docosatetraenoic acid (C22:4),docosapentaenoic acid n3 (C22:5n-3, DPAn3), and lignoceric acid (C24:0).Optionally, the four major LCFA comprise myristate, palmitic acid, DPAand DHA.

Optionally, the simple lipid profile comprises greater than 3% of eachof myristate (C14:0), palmitic acid (C16:0), docosapentaenoic acid n-6(C22:5n-6, DPAn6), and docosahexaenoic acid (C22:6n-3, DHA). Optionally,the simple lipid profile comprises triglycerides and 95% of thetriglycerides are comprised of myristate (C14:0), palmitic acid (C16:0),docosapentaenoic acid n-6 (C22:5n-6, DPAn6), and docosahexaenoic acid(C22:6n-3, DHA). Optionally, the simple lipid profile comprises lessthan 3% of each of lauric acid (C12:0), pentadecylic acid (C15:0),palmitoleic acid (C16:1), margaric acid (C17:0), stearic acid (C18:0),vaccenic acid (C18:1n-7), oleic acid (C18:1n-9), γ-linolenic acid(C18:3n-6), α-linolenic acid (C18:3n-3), stearidonic acid (C18:4),arachidic acid (C20:0), dihomo-γ-linolenic acid (C20:3n-6), arachidonicacid (C20:4n-6, ARA), eicosapentaenoic acid (C20:5n-3, EPA), behenicacid (C22:0), docosatetraenoic acid (C22:4), docosapentaenoic acid n3(C22:5n-3, DPAn3), and lignoceric acid (C24:0).

Optionally, the simple lipid profile comprises less than 0.02% shortchain fatty acids. Optionally, the simple lipid profile comprises atleast 35% C22:6n-3 (DHA) in the triglycerides in the total fatty acids.Optionally, the simple lipid profile comprises 35-40%, 35-45%, 35-50%,40-45%, 40-50%, or 50%-60% DHA in the triglycerides in the total fattyacids. Stated another way, in the triglyceride fraction of the totalfatty acids in the microorganism, the triglycerides can comprise 35-40%,35-45%, 35-50%, 40-45%, 40-50%, or 50%-60% DHA.

Optionally, the eukaryotic microorganism produces a biomass of at least20% protein, at least 40% protein, or at least 20-40% protein.

Optionally, the eukaryotic microorganism produces at least about 10%C22:6n-3 (DHA) in the triglycerides in the total fatty acids.Optionally, the eukaryotic microorganism produces at least 30% palmiticacid in the triglycerides in the total fatty acids. Optionally, theeukaryotic microorganism produces at least 40% palmitic acid in thetriglycerides in the total fatty acids. Optionally, the eukaryoticmicroorganism produces one or more carotenoids. Optionally, the one ormore carotenoids comprises β-carotene. Optionally, 0-carotene comprisesat least 95%, 96%, 97%, 98%, 99%, or 100% of the carotenoids produced inthe microorganism.

Disclosed are eukaryotic microorganisms that produce lipids, wherein theeukaryotic microorganism has a simple lipid profile. Optionally, thelipid-producing eukaryotic microorganism has an 18S sequence with atleast 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ IDNO:1. Optionally, the eukaryotic microorganism has IDAC Accession No.220716-01, which was deposited with the International DepositaryAuthority of Canada (IDAC), National Microbiology Laboratory, PublicHealth Agency of Canada, 1015 Arlington Street, Winnipeg, ManitobaCanada R3E 3R2, on Jul. 22, 2016, and assigned Accession No. 220716-01.This deposit will be maintained under the terms of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure. This deposit is exemplary and was mademerely as a convenience for those of skill in the art and is not anadmission that a deposit is required for patentability (e.g., under 35U.S.C. § 112). The terms “G3-1” or “G3-1 strain” or “strain G3-1” areused herein interchangeably to refer to the eukaryotic microorganismdeposited with the IDAC and having IDAC Accession No. 220716-01.

The provided microorganisms have distinguishing features over wild typemicroorganisms in their natural environment. Wild type microorganismscan be found in natural aquatic environments extending from oceanicenvironments to freshwater lakes and rivers, and also include brackishenvironments such as estuaries and river mouths. Such environments arenot considered to be encompassed by the term heterotrophic medium. Theprovided microorganisms produce, in a heterotrophic medium, differentamounts of one or more lipids and/or protein content from themicroorganisms in their natural environment.

Nucleic acid, as used herein, refers to deoxyribonucleotides orribonucleotides and polymers and complements thereof. The term includesdeoxyribonucleotides or ribonucleotides in either single- ordouble-stranded form. The term encompasses nucleic acids containingknown nucleotide analogs or modified backbone residues or linkages,which are synthetic, naturally occurring, and non-naturally occurring,which have similar binding properties as the reference nucleic acid, andwhich are metabolized in a manner similar to the reference nucleotides.Examples of such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). Unlessotherwise indicated, conservatively modified variants of nucleic acidsequences (e.g., degenerate codon substitutions) and complementarysequences can be used in place of a particular nucleic acid sequencerecited herein. Specifically, degenerate codon substitutions may beachieved by generating sequences in which the third position of one ormore selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al.,Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is usedinterchangeably with gene, cDNA, mRNA, oligonucleotide, andpolynucleotide.

The terms identical or percent identity, in the context of two or morenucleic acids or polypeptide sequences, refer to two or more sequencesor subsequences that are the same or have a specified percentage ofamino acid residues or nucleotides that are the same (i.e., about 60%identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or higher identity over a specified region,when compared and aligned for maximum correspondence over a comparisonwindow or designated region) as measured using a BLAST or BLAST 2.0sequence comparison algorithms with default parameters described below,or by manual alignment and visual inspection (see, e.g., NCBI web siteor the like). Such sequences are then said to be substantiallyidentical. This definition also refers to, or may be applied to, thecompliment of a test sequence. The definition also includes sequencesthat have deletions and/or additions, as well as those that havesubstitutions. As described below, the preferred algorithms can accountfor gaps and the like. Preferably, identity exists over a region that isat least about 25 amino acids or nucleotides in length, or morepreferably over a region that is 50-100 amino acids or nucleotides inlength.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A comparison window, as used herein, includes reference to a segment ofany one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981); by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970);by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988); by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.); or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

A preferred example of an algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977), and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. BLAST and BLAST 2.0 are used, with the parametersdescribed herein, to determine percent sequence identity for nucleicacids or proteins. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information, asknown in the art. This algorithm involves first identifying high scoringsequence pairs (HSPs) by identifying short words of a selected length(W) in the query sequence, which either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighborhood wordscore threshold (Altschul et al.). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are extended in both directions along each sequencefor as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. TheExpectation value (E) represents the number of different alignments withscores equivalent to or better than what is expected to occur in adatabase search by chance. The BLASTN program (for nucleotide sequences)uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5,N=−4 and a comparison of both strands. For amino acid sequences, theBLASTP program uses as defaults a wordlength of 3, expectation (E) of10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc.Natl. Acad. Sci. USA 89:10915 (1989)), alignments (B) of 50, expectation(E) of 10, M=5, N=−4, and a comparison of both strands.

The term polypeptide, as used herein, generally has its art-recognizedmeaning of a polymer of at least three amino acids and is intended toinclude peptides and proteins. However, the term is also used to referto specific functional classes of polypeptides, such as, for example,desaturases, elongases, etc. For each such class, the present disclosureprovides several examples of known sequences of such polypeptides. Thoseof ordinary skill in the art will appreciate, however, that the termpolypeptide is intended to be sufficiently general so as to encompassnot only polypeptides having the complete sequence recited herein (or ina reference or database specifically mentioned herein), but also toencompass polypeptides that represent functional fragments (i.e.,fragments retaining at least one activity) of such completepolypeptides. Moreover, those in the art understand that proteinsequences generally tolerate some substitution without destroyingactivity. Thus, any polypeptide that retains activity and shares atleast about 30-40% overall sequence identity, often greater than about50%, 60%, 70%, or 80%, and further usually including at least one regionof much higher identity, often greater than 90% or even 95%, 96%, 97%,98%, or 99% in one or more highly conserved regions, usuallyencompassing at least 3-4 and often up to 20 or more amino acids, withanother polypeptide of the same class, is encompassed within therelevant term polypeptide as used herein. Those in the art can determineother regions of similarity and/or identity by analysis of the sequencesof various polypeptides described herein. As is known by those in theart, a variety of strategies are known, and tools are available, forperforming comparisons of amino acid or nucleotide sequences in order toassess degrees of identity and/or similarity. These strategies include,for example, manual alignment, computer assisted sequence alignment andcombinations thereof. A number of algorithms (which are generallycomputer implemented) for performing sequence alignment are widelyavailable, or can be produced by one of skill in the art. Representativealgorithms include, e.g., the local homology algorithm of Smith andWaterman (Adv. Appl. Math., 1981, 2: 482); the homology alignmentalgorithm of Needleman and Wunsch (J. Mol. Biol., 1970, 48: 443); thesearch for similarity method of Pearson and Lipman (Proc. Natl. Acad.Sci. (USA), 1988, 85: 2444); and/or by computerized implementations ofthese algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package Release 7.0, Genetics Computer Group, 575Science Dr., Madison, Wis.). Readily available computer programsincorporating such algorithms include, for example, BLASTN, BLASTP,Gapped BLAST, PILEUP, CLUSTALW, etc. When utilizing BLAST and GappedBLAST programs, default parameters of the respective programs may beused. Alternatively, the practitioner may use non-default parametersdepending on his or her experimental and/or other requirements (see forexample, the Web site having URL www.ncbi.nlm.nih.gov).

The provided eukaryotic microorganisms can be cultured in aheterotrophic medium. Thus, provided herein are eukaryoticmicroorganisms having, in a heterotrophic medium, a simple lipid profilecomprising long chain fatty acids (LCFAs). Also provided are culturescomprising a lipid-producing eukaryotic microorganism with an 18Ssequence, wherein the 18S sequence has at least 98% identity to thesequence set forth in SEQ ID NO:1, and a heterotrophic medium thatresults in the lipid-producing eukaryotic microorganism having a simplelipid profile comprising long chain fatty acids (LCFAs). Optionally, thesimple lipid profile comprises greater than 3% of each of myristic acid(C14:0), palmitic acid (C16:0), docosapentaenoic acid n-6 (C22:5n-6,DPAn6), and docosahexaenoic acid (C22:6n-3, DHA). Optionally, the simplelipid profile comprises less than 3% of each of lauric acid (C12:0),pentadecylic acid (C15:0), palmitoleic acid (C16:1), margaric acid(C17:0), vaccenic acid (C18:1n-11), oleic acid (C18:1n-9), γ-linolenicacid (C18:3n-6), α-linolenic acid (C18:3n-3), stearidonic acid (C18:4),arachidic acid (C20:0), dihomo-γ-linolenic acid (C20:3n-6), arachidonicacid (C20:4n-6, ARA), (C20:3n-3), eicosapentaenoic acid (C20:5n-3, EPA),behenic acid (C22:0), docosatetraenoic acid (C22:4), docosapentaenoicacid n-3 (C22:5n-3, DPAn3), and lignoceric acid (C24:0). Optionally,95%, 96%, 97%, 98%, 99%, or 100% of the triglycerides in themicroorganism comprises myristate (C14:0), palmitic acid (C16:0),docosapentaenoic acid n-6 (C22:5n-6, DPAn6), and docosahexaenoic acid(C22:6n-3, DHA). Optionally, the simple lipid profile comprises lessthan 0.02% short chain fatty acids. Optionally, the simple lipid profilecomprises at least 35% C22:6n-3 (DHA) in the triglycerides in the totalfatty acids. Optionally, the heterotrophic medium results in productionof at least 20% protein in the whole algae biomass. Optionally, theheterotrophic medium results in production of at least 20 to 40% proteinof the biomass. Optionally, the heterotrophic medium results inproduction of at least about 40% protein. Optionally, the heterotrophicmedium further results in production of at least about 10% C22:6n-3(DHA). Optionally, the heterotrophic medium results in production of atleast 30% palmitic acid. Optionally, the heterotrophic medium results inproduction of at least 40% palmitic acid. Optionally, the heterotrophicmedium results in production of one or more carotenoids. Optionally, theone or more carotenoids comprises β-carotene, and wherein the β-carotenecomprises at least 95% of total carotenoids. Optionally, β-carotenecomprises at least 95%, 96%, 97%, 98%, 99%, or 100% of the carotenoidsproduced in the microorganism.

The provided microorganisms produce greater than 50% DHA in smallfermenters. Optionally, the provided microorganisms produce greater than50% DHA in 2 liter (L) or 5 liter (L) fermenters. Optionally, thebiomass productivity of the microorganism is from 0.5 to 0.8 g/L/h insmall fermenters, for example, 2 L or 5 L fermenters. Optionally, thetotal fatty acid productivity of the microorganism is 0.3 to 0.6 g/L/hin small fermenters, for example, 2 L or 5 L fermenters. Optionally, theDHA productivity of the microorganism is 0.1 to 0.4 g/L/h in smallfermenters, for example, 2 L or 5 L fermenters. Optionally, theproductivity of C:16 of the microorganism is 0.1 to 0.3 g/L/h in smallfermenters, for example, 2 L or 5 L fermenters.

The heterotrophic medium supplies various nutritional components,including a carbon source and a nitrogen source, for the microorganism.Medium for culture can include any of a variety of carbon sources.Examples of carbon sources include fatty acids, lipids, glycerols,triglycerols, carbohydrates, polyols, amino sugars, and any kind ofbiomass or waste stream. Fatty acids include, for example, oleic acid.Carbohydrates include, but are not limited to, glucose, cellulose,hemicellulose, fructose, dextrose, xylose, lactulose, galactose,maltotriose, maltose, lactose, glycogen, gelatin, starch (corn orwheat), acetate, m-inositol (e.g., derived from corn steep liquor),galacturonic acid (e.g., derived from pectin), L-fucose (e.g., derivedfrom galactose), gentiobiose, glucosamine, alpha-D-glucose-1-phosphate(e.g., derived from glucose), cellobiose, dextrin, alpha-cyclodextrin(e.g., derived from starch), and sucrose (e.g., from molasses). Polyolsinclude, but are not limited to, maltitol, erythritol, and adonitol.Amino sugars include, but are not limited to, N-acetyl-D-galactosamine,N-acetyl-D-glucosamine, and N-acetyl-beta-D-mannosamine. Optionally, thecarbon source is present in the heterotrophic medium at a concentrationof less than 60 g/L. Optionally, the carbon source is present in theheterotrophic medium at a concentration of 1 to 60 g/L. Optionally, thecarbon source is present in the heterotrophic medium at a concentrationof 5 to 60 g/L. Optionally, the carbon source is present in theheterotrophic medium at a concentration of 20 to 40 g/L.

Optionally, the microorganisms can be cultured in medium having achloride concentration from about 0.5 g/L to about 50.0 g/L. Optionally,microorganisms are cultured in medium having a chloride concentrationfrom about 0.5 g/L to about 35 g/L (e.g., from about 18 g/L to about 35g/L). Optionally, the microorganisms are cultured in a medium having achloride concentration from about 2 g/L to about 35 g/L. Optionally, themicroorganisms described herein can be grown in low chloride conditions.For example, the microorganisms can be cultured in a medium having achloride concentration from about 0.5 g/L to about 20 g/L (e.g., fromabout 0.5 g/L to about 15 g/L). The culture medium optionally includesNaCl. The culture medium can include non-chloride-containing sodiumsalts as a source of sodium. Examples of non-chloride sodium saltssuitable for use in accordance with the present methods include, but arenot limited to, soda ash (a mixture of sodium carbonate and sodiumoxide), sodium carbonate, sodium bicarbonate, sodium sulfate, andmixtures thereof. See, e.g., U.S. Pat. Nos. 5,340,742 and 6,607,900, theentire contents of each of which are incorporated by reference herein.Optionally, the medium comprises 9 g/L chloride when using 20 g/L ofcarbon, 20 g/L soy peptone, and 5 g/L yeast extract. Optionally, themedium comprises 35 g/L chloride when the medium contains 10 g/L carbon,5 g/L soy peptone, 5 g/L yeast extract and 10 g/L agar. Optionally, themedium comprises 2 g/L chloride when the medium contains 20-40 g/Lcarbon, 1 g/L yeast extract, 1-20 g/L monosodium glutamate (MSG),0.3-2.0 g/L phosphates, 4 g/L magnesium sulfate, 5-10 g/L ammoniumsulfate, 1.5 mL/L trace elements solution, 1 mL/L of vitamin B solution,0.1 g/L CaCl₂).

Medium for a Thraustochytrid culture can include any of a variety ofnitrogen sources. Exemplary nitrogen sources include ammonium solutions(e.g., NH₄ in H₂O), ammonium or amine salts (e.g., (NH₄)₂SO₄, (NH₄)₃PO₄,NH₄NO₃, NH₄OOCH₂CH₃ (NH₄Ac)), peptone, soy peptone, tryptone, yeastextract, malt extract, fish meal, sodium glutamate, soy extract,casamino acids and distiller grains. Concentrations of nitrogen sourcesin suitable medium typically range between and including about 1 g/L andabout 25 g/L. Optionally, the concentration of nitrogen is in the mediumis about 5 to 20 g/L. Optionally, the concentration of nitrogen in themedium is about 10 to 15 g/L. Optionally, the concentration of nitrogenin the medium is about 20 g/L. Optionally, the concentration of nitrogenis about 10 to 15 g/L when yeast extract is the source of complexnitrogen in the medium. Optionally, the concentration of nitrogen isabout 1 to 5 g/L when soy peptone is in the medium along with L-Glutamicacid monosodium salt hydrate (MSG) or ammonium sulfate.

The medium optionally includes a phosphate, such as potassium phosphateor sodium-phosphate. Optionally, the culture or heterotrophic mediumcomprises potassium phosphate monobasic.

Inorganic salts and trace nutrients in medium can include ammoniumsulfate, sodium bicarbonate, sodium orthovanadate, potassium chromate,sodium molybdate, selenous acid, nickel sulfate, copper sulfate, zincsulfate, cobalt chloride, iron chloride, manganese chloride calciumchloride, and EDTA. Optionally, the medium includes at least 1.5 ml/L ofa trace element solution. Optionally, the trace element solutioncomprises 2 mg/mL copper (II)sulfate pentahydrate, 2 mg/mL zinc sulfateheptahydrate, 1 mg/mL cobalt(II) chloride hexahydrate, 1 mg/mL manganese(II) chloride tetrahydrate, 1 mg/mL sodium molybdate dihydrate, 1 mg/mLnickel (II) sulfate.

Optionally, the medium includes magnesium sulfate. Optionally, theheterotrophic medium or culture comprises magnesium sulfate, traceelement solution and potassium phosphate monobasic.

Vitamins such as pyridoxine hydrochloride, thiamine hydrochloride,calcium pantothenate, p-aminobenzoic acid, riboflavin, nicotinic acid,biotin, folic acid and vitamin B12 can be included.

The pH of the medium can be adjusted to between and including 3.0 and10.0 using acid or base, where appropriate, and/or using the nitrogensource. Optionally, the medium can be sterilized.

Optionally, the medium comprises L-Glutamic acid monosodium salt hydrateor monosodium glutamate (MSG). Optionally, the medium comprises 1-20 g/LMSG.

Optionally, the medium comprises 1 g/L MSG when the medium comprises atleast 1 g/L yeast extract, 40 g/L carbon, 0.3 g/L KH2PO4, 4 g/Lmagnesium sulfate and 1.5 mL/L trace elements solution. Optionally, themedium comprises 20 g/L MSG when the medium comprises 5-15 g/L yeastextract, 0-10 g/L ammonium sulfate, 20-40 g/L carbon, 2 g/L chloride, 4g/L magnesium sulfate, 1.5 mL/L trace elements solution, 0.3-2.0 g/Lphosphates, 1 mL/L vitamin solution and 0.1 g/L CaCl₂.

Generally a medium used for culture of a microorganism is a liquidmedium. However, the medium used for culture of a microorganism can be asolid medium. In addition to carbon and nitrogen sources as discussedherein, a solid medium can contain one or more components (e.g., agarand/or agarose) that provide structural support and/or allow the mediumto be in solid form.

Cultivation of the microoganisms can be carried out using knownconditions, for example, those described in International PublicationNos. WO 2007/069078 and WO 2008/129358. For example, cultivation can becarried out for 1 to 30 days, 1 to 21 days, 1 to 15 days, 1 to 12 days,1 to 9 days, or 3 to 5 days. Optionally, cultivation is carried out attemperatures between 4 to 30° C. Optionally, cultivation is carried outby aeration-shaking culture, shaking culture, stationary culture, batchculture, continuous culture, rolling batch culture, wave culture, or thelike. Optionally, cultivation is carried out with a dissolved oxygencontent of the culture medium between 1 and 20%, between 1 and 10%, orbetween 1 and 5%.

Provided herein are methods of making a lipid composition. The methodsinclude culturing the provided lipid-producing eukaryotic microorganismsin a heterotrophic medium to produce a simple lipid profile andisolating the lipid composition. Optionally, the lipid-producingeukaryotic microorganism has an 18S sequence, wherein the 18S sequencehas at least 98% identity to the sequence set forth in SEQ ID NO:1, andthe heterotrophic medium results in the lipid-producing eukaryoticmicroorganism having a simple lipid profile comprising long chain fattyacids (LCFAs). Optionally, the heterotrophic medium contains less than3.75 g/L chloride. Optionally, the biomass productivity of the culturedmicororganisms is greater than 0.65 g/L/h. Optionally, the triglycerideproductivity of the cultured microorganisms is greater than 0.3 g/L/h.Optionally, the heterotrophic medium contains less than 3.75 g/Lchloride and the biomass productivity of the cultured micororganisms isgreater than 0.65 g/L/h and the triglyceride productivity of thecultured microorganisms is greater than 0.3 g/L/h. Optionally, thesimple lipid profile of the microorganism used in the methods of makinga lipid composition comprises greater than 3% of each of myristate(C14:0), palmitic acid (C16:0), docosapentaenoic acid n-6 (C22:5n-6,DPAn6), and docosahexaenoic acid (C22:6n-3, DHA). Optionally, the simplelipid profile comprises less than 3% of each of lauric acid (C12:0),pentadecylic acid (C15:0), palmitoleic acid (C16:1), margaric acid(C17:0), stearic acid (C18:0), vaccenic acid (C18:1n-7), oleic acid(C18:1n-9), γ-linolenic acid (C18:3n-6), α-linolenic acid (C18:3n-3),stearidonic acid (C18:4), arachidic acid (C20:0), dihomo-γ-linolenicacid (C20:3n-6), arachidonic acid (C20:4n-6, ARA), eicosapentaenoic acid(C20:5n-3, EPA), behenic acid (C22:0), docosatetraenoic acid (C22:4),docosapentaenoic acid n3 (C22:5n-3, DPAn3), and lignoceric acid (C24:0).

Optionally, the simple lipid profile of the microorganism used in themethods of making a lipid composition comprises less than 0.02% shortchain fatty acids. Optionally, the simple lipid profile comprises atleast 35% C22:6n-3 (DHA) in the triglycerides in the total fatty acids.Optionally, the simple lipid profile comprises 35-40%, 35-45%, 35-50%,40-45%, 40-50%, or 50-60% DHA in the triglycerides in the total fattyacids. Optionally, the eukaryotic microorganism as described hereinproduces a biomass of at least 20% protein. Optionally, the biomass isat least 20 to 40% protein. Optionally, the biomass is at least about40% protein. Optionally, the eukaryotic microorganism as describedherein produces at least about 10% C22:6n-3 (DHA) in the triglyceridesin the total fatty acids. Optionally, the eukaryotic microorganism usedaccording to the disclosed methods produces at least 30%, at least 40%,or at least 30-40% palmitic acid in the triglycerides in the total fattyacids.

Optionally, the eukaryotic microorganism produces one or morecarotenoids. Optionally, the one or more carotenoids comprisesβ-carotene. Optionally, 0-carotene comprises at least 95%, 96%, 97%,98%, 99%, or 100% of the carotenoids produced in the microorganism.

Also provided are methods of making a protein-rich biomass comprisingculturing the provided lipid-producing eukaryotic microorganisms in aheterotrophic medium and isolating the protein-rich biomass. Optionally,the method further comprises incorporating the protein-rich biomass intoa foodstuff. Optionally, the foodstuff is pet food, a livestock feed, oran aquaculture feed. Optionally, the eukaryotic microorganism as used inthe present methods produces a biomass of at least about 20% protein, atleast about 40%, or at least about 20 to 40% protein. Optionally, theeukaryotic microorganism produces at least about 10% C22:6n-3 (DHA) inthe triglycerides in the total fatty acids.

Optionally, the lipids produced according to the methods describedherein can be incorporated into a final product (e.g., a food or feedsupplement, an infant formula, a pharmaceutical, a fuel, and the like).Thus, provided is a method of using the lipid composition made accordingto the methods described herein, wherein the method of use comprisesincorporating the lipid composition into a foodstuff.

Further, the provided protein-rich biomass can be incorporated into afinal product (e.g., food or feed supplement, biofuel, etc.). Thus,provided is a method of using the protein-rich biomass comprisingincorporating the protein-rich biomass into a foodstuff (e.g., a petfood, a livestock feed, or an aquaculture feed).

Suitable food or feed supplements into which the lipids can beincorporated include beverages such as milk, water, sports drinks,energy drinks, teas, and juices; confections such as candies, jellies,and biscuits; fat-containing foods and beverages such as dairy products;processed food products such as soft rice (or porridge); infantformulae; breakfast cereals; or the like. Optionally, one or moreproduced lipids can be incorporated into a dietary supplement, such as,for example, a vitamin or multivitamin. Optionally, a lipid producedaccording to the method described herein can be included in a dietarysupplement and optionally can be directly incorporated into a componentof food or feed (e.g., a food supplement).

Examples of feedstuffs into which lipids produced by the methodsdescribed herein can be incorporated include pet foods such as catfoods; dog foods; feeds for aquarium fish, cultured fish or crustaceans,etc.; feed for farm-raised animals (including livestock and fish orcrustaceans raised in aquaculture). Food or feed material into which thelipids produced according to the methods described herein can beincorporated is preferably palatable to the organism which is theintended recipient. This food or feed material can have any physicalproperties currently known for a food material (e.g., solid, liquid,soft).

Optionally, one or more of the produced compounds (e.g., PUFAs) can beincorporated into a nutraceutical or pharmaceutical product. Examples ofsuch a nutraceuticals or pharmaceuticals include various types oftablets, capsules, drinkable agents, etc. Optionally, the nutraceuticalor pharmaceutical is suitable for topical application. Dosage forms caninclude, for example, capsules, oils, granula, granula subtilae,pulveres, tabellae, pilulae, trochisci, or the like.

The oil or lipids produced according to the methods described herein canbe incorporated into products in combination with any of a variety ofother agents. For instance, such compounds can be combined with one ormore binders or fillers, chelating agents, pigments, salts, surfactants,moisturizers, viscosity modifiers, thickeners, emollients, fragrances,preservatives, etc., or any combination thereof.

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed methods and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutations of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. For example, if a method is disclosed and discussed and a numberof modifications that can be made to a number of molecules including themethod are discussed, each and every combination and permutation of themethod, and the modifications that are possible are specificallycontemplated unless specifically indicated to the contrary. Likewise,any subset or combination of these is also specifically contemplated anddisclosed. This concept applies to all aspects of this disclosureincluding, but not limited to, steps in methods using the disclosedcompositions. Thus, if there are a variety of additional steps that canbe performed, it is understood that each of these additional steps canbe performed with any specific method steps or combination of methodsteps of the disclosed methods, and that each such combination or subsetof combinations is specifically contemplated and should be considereddisclosed.

Publications cited herein and the material for which they are cited arehereby specifically incorporated by reference in their entireties.

The examples below are intended to further illustrate certain aspects ofthe methods and compositions described herein, and are not intended tolimit the scope of the claims.

EXAMPLES Example 1. Identification and Preliminary Analysis ofThraustochytrid-Like Strain G3-1

After two days of incubation strain G3-1 showed significant accumulationof biomass. In contrast, other thrasutochytrids, under the conditionsemployed, required at least three days of incubation to achieve asimilar level of biomass accumulation. Preliminary analysis alsoindicated that thraustochytrid-like strain G3-1 was able to accumulatehigh concentrations of oil rich in docosahexaenoic acid (DHA) andpalmitic acid (C16:0). DHA is a fatty acid of high value due to use inhuman and animal nutrition. Palmitic acid is also of value when employedas a feedstock for biofuel production. Furthermore, with the applicationof animal nutrition in mind and aquaculture in particularthraustochytrid-like strain G3-1 has been shown to accumulate protein toa level that composes around 30% of its biomass dry weight. Due to theseproperties thraustochytrid-like strain G3-1 was selected to develop amethod to produce biomass rich in DHA, palmitic acid and protein in ashort period of time.

Example 2. Evaluation of Strain G3-1 in Two Media Compositions

To evaluate the thraustochytrid-like strain G3-1, it was cultivatedunder two conditions, full fermentation (FF) media and Basal (B) media.FF media is a complex media and B media is a minimal media used in theanalysis and development of different processes, strains and conditions.

A comparison of G3-1 productivity in FF vs B media demonstrated thatbiomass content was higher in B media with G3-1 producing 53% morebiomass (Table 1). Also total fatty acid (TFA) yield increased on avolumetric basis by 49%, owed to higher biomass accumulation. Moreover,on a dry cell weight basis TFA production also increased. In FF mediafatty acids composed 289 mg g⁻¹ biomass while in B media this increasedto 312 mg g⁻¹ biomass, an increase of 8% (Table 1). The obtained resultssuggest that strain G3-1 may be sensitive to the osmotic pressureimposed high concentration of glucose in FF media. Protein content wasalso analyzed revealing that up to 30% of dry cell weight is attributedto protein.

Experimental Details

Growth and cultivation—Seed cultures were produced by either adding 1 mLof the ASW containing pure strain G3-1 culture or two loops of purecolonies taken from an agar plate, to 30 mL aliquots of B media in 150mL baffled Erlenmeyer flasks. Flasks were incubated at 25° C. and 200rpm for 2 days. 5 mL aliquots of seed cultures (previously adjusted withsterile fresh B media to OD600 nm=1.5) were taken under asepticconditions and added to 95 mL of sterile test media in 500 mL Erlenmeyerflasks. Test media composition and culture conditions for these flaskswere evaluated using a B media (described above) and FF media, which wascomposed of 60 g L⁻¹ glucose, 2 g L⁻¹ sea salt, 4 g L⁻¹ soy peptone, 1 gL⁻¹ yeast extract, 4 g L⁻¹ magnesium sulfate, 2 g L⁻¹ sodium chloride, 5mg L⁻¹ ferric chloride, 3 mg L⁻¹ copper sulfate, 2 mg L⁻¹ sodiummolybdate, 3 mg L⁻¹ zinc sulfate, 2 mg L⁻¹ cobalt (II) chloride, 2 mgL⁻¹ manganese chloride, 2 mg L⁻¹ nickel sulfate, 1.6 g L⁻¹ potassiumphosphate monobasic, 1.75 g L⁻¹ potassium phosphate dibasic, 6.8 g L⁻¹ammonium sulfate, 0.1 g L⁻¹ calcium chloride dehydrate, 0.01 g L⁻¹cobalamin, 0.01 g L⁻¹ biotin and 2 g L⁻¹ thiamin hydrochloride. Aftertwo days of fermentation, broth samples were taken from each flask andcells were harvested by centrifugation at 4150 rpm for 20 min at 2° C.The pellet was rinsed with distilled water to remove the salts andresidual substrate, and then re-centrifuged. Pellets were frozen at −80°C., freeze-dried and stored at −20° C. prior to biomass and fatty acidanalysis. The freeze-dried cell pellets were weighed to determine thebiomass of strain G3-1 culture and reported as dry weight of cells perunit volume of media (g L⁻¹). A direct one step transesterificationmethod was carried out to prepare fatty acid methyl esters (FAMEs) fromfreeze-dried biomass to estimate the oil content inside the cells. FIG.1 demonstrates that media composition had a significant effect (p<0.05)on biomass and TFA production by G3-1. See also Table 1.

TABLE 1 Biomass and TFA production by strain G3-1 in liquid media. MediaBiomass (g L⁻¹) TFA (g L⁻¹) Full  6.9 ± 0.3 2.0 ± 0.1 fermentation (FF)Basal (B) 13.1 ± 0.3 4.1 ± 0.3

This investigation also demonstrated that media composition was able tomodify the fatty acid profile of the oil produced by G3-1 (FIG. 2). WhenG3-1 was cultured in B media, the composition of fatty acids slightlychanged compared to FF media. However, DHA was still produced at high aconcentration, G3-1 produced DHA at 47.1% and 46.9% of total fatty acidcontent, when cultured in FF and B media, respectively. See Table 2.

TABLE 2 Effect of media composition on fatty acid profile of theintracellular oil of strain G3-1: basal (B), full fermentation media(FF). Fatty FF B FF B acids mg/g mg/g % % C14:0  6.4 ± 0.4  6.6 ± 0.4 2.2 ± 0.0  2.1 ± 0.0 C15:0  2.3 ± 0.1  47.1 ± 0.7  0.8 ± 0.1 15.1 ± 0.9C16:0 105.2 ± 6.6  63.1 ± 4.5 36.7 ± 0.3 20.1 ± 0.6 C17:0  1.0 ± 0.1 9.5 ± 0.1  0.4 ± 0.0  3.0 ± 0.1 C18:0  3.2 ± 0.2  1.9 ± 0.1  1.1 ± 0.0 0.6 ± 0.0 C20:4-6  1.2 ± 0.3  2.2 ± 0.1  0.4 ± 0.1  0.7 ± 0.0 EPA  1.1± 0.3  0.8 ± 0.0  0.4 ± 0.1  0.3 ± 0.0 C22:5  29.8 ± 1.9  31.0 ± 1.410.4 ± 0.1  9.9 ± 0.0 n-6 DPA DHA 134.9 ± 9.4 146.9 ± 7.4 47.1 ± 0.246.9 ± 0.3 TFA 285.1 309.1

To analyze the protein content of strain G3-1 biomass a hot-TCA methodwas adapted from the literature and optimized using G3-1 culture aged at16 h and 22 h. Four combinations of hot-TCA conditions were evaluatedand the resultant protein content is shown in FIG. 3. Using condition 4over 30% protein content was detected in G3-1, revealing the potentialof this strains to produce meaningful amounts of protein (i.e., >40%)and therefore could serve as a partial fish meal replacement product.

Example 3. Nutritional Requirements for Enhanced Biomass Production andLipid Accumulation by Thraustochytrid-Like Strain G3-1

Previous analysis comparing FF and B media demonstrated that thenutritional requirements of G3-1 differ to those of otherthrasutochytrids. A series of experiments were designed to betterunderstand what aspects of B media affect growth and lipid productionfor G3-1. To do this, a standard factorial design experimental approachwas applied, also called a Plackett-Burman.

The results show that the nutrient requirements of strain G3-1 differfrom those of other thraustochytrids, that G3-1 has the capacity toproduce short chain saturated fatty acids (palmitic acid) and long chainpolyunsaturated fatty acids (DHA), likely through independent pathways,a classic elongation and desaturation pathway plus an independentpolyketide synthase pathway. They also demonstrate that soy peptone andsea salt together have a significant impact on G3-1 productivity.

Experimental Details

An irregular fraction factorial design (2{circumflex over ( )}4*3/4) wasused to explore the significance of four independent variables, B mediacomponents, on biomass and fatty acid production. Independent variableswere tested at a high (+1) and low (−1) concentrations (Table 3). Atotal of twelve experimental runs were completed in duplicate, asdescribed in Table 4.

TABLE 3 Independent variables and their levels used in the irregularfraction factorial design. Coded level Variables Coded Xi −1 +1 Glucose(g L⁻¹) X1 20 40 Soy peptone (g L⁻¹) X2  4 20 Yeast extract (g L⁻¹) X3 1  5 Sea salt (g L⁻¹) X4  2  9

TABLE 4 Irregular fraction factorial design matrix. Coded VariableProcess Variable Run Block X1 X2 X3 X4 X1 X2 X3 X4  1 1 −1 −1 −1 −1 20 41 2  2 1 −1 −1 −1 +1 20 4 1 9  3 1 −1 −1 +1 −1 20 4 5 2  4 1 −1 −1 +1 +120 4 5 9  5 1 −1 +1 −1 −1 20 20 1 2  6 1 −1 +1 −1 +1 20 20 1 9  7 1 −1+1 +1 −1 20 20 5 2  8 1 −1 +1 +1 +1 20 20 5 9  9 1 +1 −1 −1 +1 40 4 1 910 1 +1 −1 +1 −1 40 4 5 2 11 1 +1 +1 −1 −1 40 20 1 2 12 1 +1 +1 +1 +1 4020 5 9

interaction between soy peptone and sea salt concentration (X2*X4) had asignificant effect (p<0.05) on the ability of G3-1 to produce biomass.Additionally, the amount of intracellular fatty acids accumulated bystrain G3-1 was significantly (p<0.05) affected by soy peptone (X2),yeast extract (X3) and sea salt concentration (X4), and the interactionbetween glucose and sea salt concentration (X1*X4). In addition toaffecting biomass and lipid productivity, fatty acid profile was alsoinfluenced by the changes imposed (FIG. 4). Broadly speaking, highamounts of DHA, 47.7 to 51.0% of TFA, were synthesized by G3-1 cells inthis experiment for the following the combination of ingredients forruns 6, 7, 8 and 12 (in Table 4). Thus, G3-1 has the metabolic capacityto easily produce biomass composed of >50% DHA. Equally important, thisseries of experiments identified conditions that result in theproduction of lower amounts of saturated fatty acids, and affectedoverall productivity (Table 5, FIG. 4). The composition of the liquidmedia selected to increase biomass production, TFA yield and DHA bystrain G3-1 at a lab scale was 20 g L⁻¹ glucose, 20 g L⁻¹ soy peptone, 5g L⁻¹ yeast extract and 9 g L⁻¹ sea salts. This is the same as thepreviously described Basal (B) media.

TABLE 5 Fatty acid profile of the intracellular oil of strain G3-1 underthe different liquid media compositions tested using an irregularfraction factorial design. Fatty Acids (%) C18:1 C18:1 C20:4 Run C14:0C15:0 C16:0 C16:1 C17:0 C18:0 Ole Vac C20:0 n-6  1 2.9 6 41.1 0.3 1.71.1 0.1 0.4 0.2 1.7  2 3.6 5.1 39.7 0.3 1.3 1.1 0.1 0.4 0.3 1.6  3 3 6.737.3 0.3 1.6 1 0.2 0.5 0.2 1.4  4 3.9 12 30.5 0.3 2.3 0.9 0.2 0.5 0.21.4  5 1.7 29.1 11.1 0.2 4.6 0.3 0.3 0.3 0 1  6 1.2 20.5 12.3 0.2 5 0.30.5 1.1 0 1.3  7 1 18.4 11.9 0 4.5 0.1 0.7 0.9 0 1.4  8 1.1 17 12.9 04.8 0.2 0.6 1.2 0 1.4  9 3.5 4.2 40.9 0.2 1.1 1.1 0.1 0.3 0.2 1.1 10 2.826.9 15.1 0.4 4.4 0.4 0.4 0.9 0 1.7 11 1.9 21.9 11.9 0.5 4.3 0.4 0.7 1.10 1.7 12 1.2 18 15.2 0 5 0.5 0.3 0.5 0 0.9 Fatty Acids (%) C22:5 n-6Biomass TFA Run EPA DPA DHA SFA MUFA PUFA (g/L) (g/L/d)  1 0.5 4.5 38.753 0.8 45.4 5.1 ± 0.09 2.26 ± 0.08  2 0.5 5.6 40.2 51.1 0.8 47.9 5.7 ±0.12 2.59 ± 0.04  3 0.4 5.7 41 49.8 1 48.5 5.3 ± 1.00 1.82 ± 0.34  4 0.56 40.6 49.8 1 48.5 5.9 ± 0.12 1.89 ± 0.06  5 0.4 7.7 42.1 46.8 0.8 51.27.6 ± 0.43 1.27 ± 0.06  6 0.6 8.3 47.8 39.3 1.8 58 6.0 ± 0.50 0.64 ±0.08  7 0.6 8.7 51 35.9 1.6 61.7 7.2 ± 0.85 0.71 ± 0.04  8 0.7 8.9 50.536 1.8 61.5 6.6 ± 1.05 0.69 ± 0.11  9 0.4 6.5 40 51 0.6 48 5.8 ± 0.693.02 ± 0.47 10 0.8 4.6 40.4 49.6 1.7 47.5 5.0 ± 0.66 0.90 ± 0.13 11 0.86.9 46 40.4 2.3 55.4 6.8 ± 0.81 0.68 ± 0.07 12 0.5 9.1 47.7 39.9 0.858.2 7.8 ± 0.27 0.90 ± 0.02

Example 4. Evaluation of Thraustochytrid-Like Strain G3-1 in LabFermenters

Basal (B) media formulation from run 8, described above, was selected tocarry out fermentations using 2 L fermenters to evaluate the potentialof the strain G3-1 to produce biomass rich in DHA and palmitic acid.

In an 88 hour fermentation thraustochytrid-like strain G3-1 produced51.8 g L⁻¹ biomass composed of 67.3% TFA. DHA and palmitic acidconstituted 38.8% and 44.7% of TFA, respectively. TFA productivity was0.398 g L⁻¹ hr⁻¹ which exceeded published examples by >32%.

Experimental Details

G3-1 was pre-cultured in Erlenmeyer flasks containing 500 mL of liquidmedia (20 g L⁻¹ glucose, 20 g L⁻¹ soy peptone, 5 g L⁻¹ yeast extract and9 g L⁻¹ sea salts). Flasks were incubated under agitation at 25° C. and200 rpm for 2 days. After the incubation period, 200 mL of thepre-culture was transferred into 1.8 L of the same media, in a 2-Lfermentation vessel. Batch culture conditions were applied as follows:25° C., agitation starting at 400 rpm and reaching 600 rpm, aeriation at0.3 VVM with atmospheric air, and pH 6.8. Cells were collected at 10-15h intervals and growth, oil (TFA) and DHA content examined.

Glucose in the media was completely consumed after 20 h of fermentation.Fed-batch cultures were carried out for a total of 88 h. After 65 h offed-batch fermentation, G3-1 produced 34.7 g L⁻¹ of biomass and 77.1%TFA. DHA (40.6%) and palmitic acid (42.7%) where the main fatty acidsfound in the lipid produced by this strain. After 88 h of fermentation,the total biomass accumulated was 51.8 g L⁻¹. At the end of thefermentation, 88 hours, dry biomass, TFA and DHA measured 51.8 g L⁻¹,70% and 38.8%, respectively. Thus, for this fermentation total fattyacid productivity was 0.396 g L⁻¹ h⁻¹ and DHA productivity was 0.154 gL⁻¹ h⁻¹. Table 6 and FIG. 5 presents the fatty acid profile of G3-1 whencultured under the described fermentation conditions. As a startingpoint with minimal process optimization G3-1 is considered a very highDHA production strain.

TABLE 6 Oil production and fatty acid profile of the intracellular oilof strain G3-1. Cells were cultured in 2-L fermenters using a selectedbasal media. Fatty acids (%) C20:5 C22:5 C22:6 Time TFA (n-3) (n-6)(n-3) Biomass TFA Productivity (g L⁻¹ h⁻¹) (h) (%) C14:0 C15:0 C16:0C18:0 EPA DPA DHA (g/L) (mg/g) Biomass TFA DHA C16:0 Seed 19.6 2.8 10.626.3 0.9 1.7 5.6 44.3 13.1 196.3 21.34 24.0 2.7 15.5 25.7 0.8 0.5 7.942.4 14.7 240.2 40.28 49.5 2.9 3.9 35.8 1.0 0.3 8.4 45.5 28.1 495.4 4755.6 3.1 3.1 38.3 1.1 0.3 8.2 43.9 34.8 555.7 65 77.1 3.8 2.1 42.7 1.10.3 7.7 40.6 34.7 770.9 71 74.2 3.9 2.0 44.1 1.1 0.3 7.5 39.5 36.9 742.088 67.3 3.9 2.0 44.7 1.1 0.4 7.4 38.8 51.8 672.6 0.589 0.396 0.154 0.177

Example 5. Improving the Process Through the Use of a Modified FullFermentation Media

In order to achieve higher biomass productivity, the effect of amodified full fermentation (MFF) media was assessed for impact onbiomass and fatty acid accumulation with respect to strain G3-1. Themodified fermentation (MFF) media is a complex media rich in mineralsand vitamins that is identical to that employed previously, i.e., FF,except that glucose was reduced from 60 g L⁻¹ to 20 g L⁻¹ to reduceosmotic pressure. 2 L fed-batch fermentations were carried out andchanges in biomass and fatty acid content monitored. In this 115 hourfermentation thraustochytrid-like strain G3-1 produced 85.4 g L⁻¹biomass composed of 63.6% TFA. DHA and palmitic acid constituted 43.4%and 41.0% of TFA, respectively. TFA productivity was 0.471 g L⁻¹ h⁻¹,which exceeded published examples by >56%.

Experimental Details

A modified full fermentation media (MFF) was used to culture strain G3-1in a 2-L fermenter for enhanced biomass and fatty acid production. G3-1was pre-cultured in Erlenmeyer flasks containing 500 mL of the selectedbasal media (20 g L⁻¹ glucose, 20 g L⁻¹ soy peptone, 5 g L⁻¹ yeastextract and 9 g L⁻¹ sea salts). Flasks were incubated under agitation at25° C. and 200 rpm for 2 days. After the incubation period, 200 mL ofthe pre-cultured cells were transferred into 1.8 L of MFF media (20 gL⁻¹ glucose, 2 g L⁻¹ sea salt, 4 g L⁻¹ soy peptone, 1 g L⁻¹ yeastextract, 4 g L⁻¹ magnesium sulfate, 2 g L⁻¹ sodium chloride, 5 mg L⁻¹ferric chloride, 3 mg L⁻¹ copper sulfate, 2 mg L⁻¹ sodium molybdate, 3mg L⁻¹ zinc sulfate, 2 mg L⁻¹ cobalt (II) chloride, 2 mg L⁻¹ manganesechloride, 2 mg L⁻¹ nickel sulfate, 1.6 g L⁻¹ potassium phosphatemonobasic, 1.75 g L⁻¹ potassium phosphate dibasic, 6.8 g L⁻¹ ammoniumsulfate, 0.1 g L⁻¹ calcium chloride dehydrate, 0.01 g L⁻¹ cobalamin,0.01 g L⁻¹ biotin and 2 g L⁻¹ thiamin hydrochloride) and batch culturedin 2-L fermenters under the conditions of 25° C., agitation starting at450 rpm and reaching 500 rpm, aeriation at 0.3 VVM with atmospheric air,and pH 6.8. Cells were collected at 10-15 hr intervals and the biomass,TFA and DHA were measured. Glucose in the media was completely consumedafter 18-20 h of fermentation and at that time. The culture was thenbatch fed with 75% (w/v) glucose, until 115.38 h when the fermentationwas ended. At the end of the fermentation (115.38 h) biomass, TFA andDHA measured 85.4 g L⁻¹, 63.6% and 43.4% DHA (Table 7, FIG. 6). For thisfermentation productivity for biomass, TFA and DHA was 0.740 g L⁻¹ h⁻¹,0.471 g L⁻¹ h⁻¹ and 0.204 g L⁻¹ h⁻¹, respectively. The oil obtainedunder these conditions was high in DHA.

TABLE 7 Oil production and fatty acid profile of the intracellular oilof MARA G3-1. Cells were cultured in 2-L fermenters using a modifiedfull fermentation media. Fatty Acids (%) C20:5 C22:5 C22:6 Time TFA(n-3) (n-6) (n-3) Biomass TFA Productivity (g/L/h) (h) (%) C14:0 C15:0C16:0 C18:0 EPA DPA DHA (g/L) (mg/g) Biomass TFA DHA C16:0 18.25 13.81.7 2.0 30.2 0.8 0.9 10.1 52.2 16.6 137.5 40.36 46.2 3.4 0.5 37.6 1.00.4 9.1 46.7 42.0 462.3 46.22 50.1 3.4 0.4 37.4 1.0 0.3 9.0 47.0 49.3501.1 70.43 60.3 3.8 0.3 38.7 1.0 0.4 8.7 45.6 67.5 603.1 96.45 66.0 4.00.3 39.4 1.0 0.4 8.3 45.0 80.4 659.9 115.38 63.6 4.2 0.3 41.0 1.0 0.48.1 43.4 85.4 636.3 0.740 0.471 0.204 0.193

Biomass production was successfully enhanced when an MFF media was usedto culture G3-1 in 2 L-fermenters. However, after 115.38 h of fed-batchfermentation TFA reached 63.6% of biomass dry weight. A possibleexplanation for this is that MFF contained less soy peptone and sea saltthan B media. Components that were found to be important in the initialfactorial design experiment.

Example 6. The Effect of Nitrogen Limitation on the Oil Accumulation byThraustochytrid-Like Strain G3-1

The modified full fermentation media (MFF), described above, wasadjusted to reduce the concentration of inorganic nitrogen (in the formof ammonium sulphate) in the liquid media by 50%. Strain G3-1 wascultured in 2 L-fermenters. Glucose consumption was monitored and, whenthe cells showed signs of starvation, fermentations were fed-batch with75% (w/v) glucose.

In this 112 hour fermentation, thraustochytrid-like strain G3-1 produced79.5 g L⁻¹ biomass composed of 77.4% TFA. DHA and palmitic acidconstituted 36.9% and 48.2% of TFA, respectively. TFA productivity was0.548 g L⁻¹ h⁻¹, which exceeds published examples by >82%.

Experimental Details

G3-1 was pre-cultured following the same conditions describedpreviously. 200 mL of pre-cultured cells were transferred to 1.8 L MFFmedia with half the normal amount of inorganic nitrogen (2-L vesselfermenter). Media was formulated using 3.4 g L⁻¹ of ammonium sulphateand all the other ingredients were maintained at the same concentrationas described previously. Culture conditions were 25° C., agitationstarted at 480 rpm and increased 500 rpm over the course of thefermentation, aeriation was maintained at 0.3 VVM with atmospheric air,and pH 6.8 was maintained. Cells were collected at 10-15 hr intervalsand biomass, TFA and DHA contents were analysed.

FIG. 7 shows that using half of the concentration of ammonium sulphatein MFF media accelerated the rate of palmitic acid (C16:0) production bythe employed strain. At 40 h, nitrogen was exhausted. The data in Table8 shows that both biomass and TFA continued to accumulate in the G3-1culture. It is also apparent that little change in the TFA profileoccurred after nitrogen limitation (FIG. 7).

In this investigation, TFA increased from 18.3 to 75.9% (maximally) ofdry cell weight, as a percentage of TFA palmitic acid increased fromaround 45% to 48% over the course of the fermentation. Over the sametime period DHA reduced slightly from 45% to 36.9% (Table 8). Thiscorresponds to the biosynthesis of 372.7 mg g⁻¹ palmitic acid and 285.3mg g⁻¹ DHA. Production rate for these two fatty acids is 0.264 g L⁻¹ h⁻¹palmitic acid and 0.202 g L⁻¹ h⁻¹ DHA. The observed increase in palmiticacid biosynthesis is not surprising because nitrogen stress is known toinduce the expression of genes in the classic fatty acid synthesispathway responsible for palmitic acid production. However, it isreassuring to see that DHA production remains approximately the sameirrespective of nitrogen stress.

TABLE 8 Oil production and fatty acid profile of the intracellular oilof strain G3-1. Cells were cultured in 2-L fermenters using halfconcentration of ammonium sulphate in a modified full fermentationmedia. Fatty acids (%) C20:5 C22:5 C22:6 Time TFA (n-3) (n-6) (n-3)Biomass TFA Productivity (g/L/h) (h) (%) C14:0 C15:0 C16:0 C18:0 EPA DPADHA (g/L) (mg/g) Biomass TFA DHA C16:0 16 18.3 2.6 1.2 37.1 1.0 0.7 9.045.2 16.41 183.5 23.16 37.7 3.4 0.7 46.0 1.2 0.4 7.7 38.7 23.7 376.940.00 59.0 4.8 0.4 45.2 1.1 0.3 7.2 38.7 37.8 589.8 47.07 62.5 4.9 0.445.2 1.1 0.3 7.2 38.8 44.0 624.6 64.18 70.1 5.0 0.3 45.5 1.1 0.3 7.238.6 55.9 700.8 70.15 73.1 4.8 0.3 46.0 1.1 0.3 7.1 38.2 58.5 730.988.09 73.8 4.8 0.2 46.8 1.1 0.3 7.1 37.7 70.0 738.0 94.45 75.9 4.7 0.247.0 1.1 0.3 7.1 37.6 73.5 759.1 112.19 77.4 4.6 0.2 48.2 1.1 0.3 7.036.9 79.5 773.5 0.709 0.548 0.202 0.264

Example 7. Taxonomic Characterization of the Thraustochytrid-Like StrainG3-1

A standard approach to characterize strain G3-1 was applied. The 18SrDNA sequence was amplified from G3-1 genomic DNA using Taq DNApolymerase and primers JBo119 (5′-CAACCTGGTTGATCCTGCCAGTA-3′ (SEQ IDNO:2)) and JBo120(5′-TCACTACGGAAACCTTGTTACGAC-3′ (SEQ ID NO:3)). The 50μl PCR reaction contained 1.25 Units Taq DNA polymerase (New EnglandBiolabs, M0273 (IpSwich, MA)), lx standard reaction buffer, 200 μM ofeach dNTP (A,G,C,T), 0.2 μM of each primer (JBo119 and JBo120), and 3%DMSO. This PCR reaction was incubated at 95° C. for 4 min., thensubjected to 35 cycles of 95° C. for 30 sec., 52° C. for 30 sec., and68° C. for 1:45 min., followed by a final incubation at 68° C. for 30min., before being held at 4° C. The ˜1.7 kb amplicon was gel purifiedand this pool of amplified fragments was cloned into pCR2.1 vector by TAcloning to produce the plasmid pJB84. Ten individual clones of pJB84(#1, 2, 5, 6, 7, 10, 12, 13, 14, and 15) were isolated and sent toGenewiz (South Plainfield, N.J.) for sequencing. The 18S rRNA sequenceof each clone are provided in SEQ ID NOs:10, 11, 12, 13, 14, 15, 16, 17,18, and 19. Each clone was sequenced with 8 primers in total: 4 forwardprimers and 4 reverse primers. Two of the sequencing primers, M13R andT7, are universal primers that bind vector sequences flanking the TAcloning site and were provided by Genewiz. The other 6 primers,including JBo119 and JBo120 used to amplify the 18S rDNA, bind withinthe 18S rDNA sequence and were designed based on previously reportedprimer sequences (Burja, A. M., Radianingtyas, H., Windust, A., andBarrow, C. J. (2006). Isolation and characterization of polyunsaturatedfatty acid producing Thraustochytrium species: screening of strains andoptimization of omega-3 production. Appl. Microbiol. Biotechnol. 72,1161-1169; Mo, C., J., D., and B., R. (2002). Development of a PCRstrategy for thraustochytrid identification based on 18S rDNA sequence.Mar. Biol. 140, 883-889). The 8 primer sequences are:

M13R (SEQ ID NO: 4) 5′-CAG GAA ACA GCT ATG AC-3′ (universal primer) T7(SEQ ID NO: 5) 5′-TAA TAC GAC TCA CTA TAG GG-3′ (universal primer)JBo119 (SEQ ID NO: 2) 5′-CAACCTGGTTGATCCTGCCAGTA-3′ (Burja et al. 2006)JBo120 (SEQ ID NO: 3) 5′-TCACTACGGAAACCTTGTTACGAC-3′ (Burja et al. 2006)JBo121 (SEQ ID NO: 6) 5′-GTCTGGTGCCAGCAGCCGCG-3′ (Mo et al. 2002) JBo122(SEQ ID NO: 7) 5′-CTTAAAGGAATTGACGGAAG-3′ (Mo etal. 2002) JBo123(SEQ ID NO: 8) 5′-AGCTTTTTAACTGCAACAAC-3′ (Mo etal. 2002) JBo124(SEQ ID NO: 9) 5′-GGCCATGCACCACCACCC-3′ (Mo etal. 2002)

The 8 sequencing reactions for each clone were trimmed by deleting the5′ and 3′ sequences containing N's (ambiguous nucleotides) leaving onlythe successful portions of each sequencing read. These were assembledinto a single contig for each clone using ChromasPro software(Technelysium Pty Ltd, South Brisbane, Australia). These contigscontained the 18S rDNA sequences as well as flanking vector sequences.The vector sequences were trimmed from the contig leaving only the 18SrDNA sequence amplified by JBo119 and JBo120. Any ambiguous nucleotidesindicated by ChromasPro were manually determined, but there were veryfew, if any of these. In all cases the 18S rDNA sequence was covered byat least 2 sequencing reads over its entire length, but had at least 3reads coverage for the vast majority of its length, with >3 readscovering some shorter spans. All 10 sequences were at least 98%identical to each other. The largest variability between any pair ofclones is between #5 and #6 which are 98.19% identical. There are twopairs of clones that are 100% identical, #1 and 7, and #12 and 13, andthese identical pairs are 98.98% identical to each other. A consensussequence was created. G3-118S rDNA consensus sequence based on 10individually sequenced clones is shown below. Degenerate nucleotideswere manually curated using the standard IUPAC annotation (A, Adenine;C, Cytosine; G, Guanine; T, Thyamine; W, A or T; S, C or G; M, A or C;K, G or T; R, A or G; Y, C or T; B, not A; D, not C; H, not G; V, not T;N, any Nucleotide).

(SEQ ID NO: 1) CAACCTGGTTGATCCTGCCAGTAGTCATATGCTCGTCTCAAAGATTAAGCCRTGCATGTGTAAGTATAAG CGATTGTACTGTGAGACTGCGAACGGCTCATTATATCAGTAATAATTWCTTCGGTARYTTCTTTTATATG GATACCTGCAGTAATTCTGGAAATAATACATGCTGTAAGAGCCCTRTATGGGGCTGCACTTATTAGATTG AAGCCGATTTTATTGGTGAATCATGATAATTGAGCAGATTGACTWTTTTTDGTCGATGAATCGTTTGAGT TTCTGCCCCATCAGTTGTCGACGGTAGTGTATTGGACTACGGTGACTATAACGGGTGACGGAGAGTTAGG GCTCGACTCCGGAGAGGGAGCCTGAGAGACGGCTACCATATCCAAGGATAGCAGCAGGCGCGTAAATTAC CCACTGTGGACTCCACGAGGTAGTGACGAGAARYATCGATGCGAAGCGTGTATGCGTTTTGCTATCGGAA TGAGARYAATGTAAAACCCTCATCGAGGATCAACTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTRATT CCAGCTCCRGAAGCATATGCTAAAGTTGTTGCAGTTAAAAAGCTCGTAGTTGAATTTCTGGCATGGGCGA CCGGTGCTTTCCCTGAATGGGGATWGATTGTCTGTGTTGCCTTGGCCATCTTTYTCWTKYYDTTWTWGRK RWGARATCTTTCACTGTAATCAAAGCAGAGTGTTCCAAGCAGGTCGTATGACCGGTATGTTTATTATGGG ATGATAAGATAGGACTTGGGTGCTATTTTGTYGGTTTGCACGCCTGAGTAATGGTTAATAGGAACAGTTG GGGGTATTCGTATTTAGGAGCTAGAGGTGAAATTCTTGGATTTCCGAAAGACGAACTAGAGCGAAGGCAT TTACMAAGCATGTTYTCATTAATCAAGAACGAAAGTCTGGGGATCGAAGATGATTAGATACCATCGTAGT CTAGACCGTAAACGATGCCRACTTGCGATTGTTGGGTGCTTTWTTDTATGGGCCTCAGCAGCRGCACATG AGARATCAAAGTCTTTGGGTTCCGGGGGGAGTATGGTCGCAAGGCTGAAACTTRAAGGAATTGACGGAAG GGCACCACCAGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGAAAACTTACCAGGTCCAGACATA GGTAGGATTGACAGATTGAGAGCTCTTTCATGATTCTATGGGTGGTRGTGCATGGCCKTTCTTAGTTGGT GGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTCGGCCTACTAAATAGTGCGTGGTATGGCA ACATAGTRCGTTTTWAACTTCTTAGAGGGACATGTCCGGTTTACGGGCAGGAAGTTCGAGGCAATAACAG GTCYGTGATGCCCTTAGATGYTCTGGGCCGCACGCGCGCTACACTGATGGGTTCATCGGGTTTTRATTYY AWTTWWTGGAATTGAGTGCTTGGTCGGAAGGCCTGGCTAATCCTTGGAACGCTCATCGYGCTGGGGCTAG ATTTTYGCAATTATTAATCTCCRACGAGGAATTCCTAGTAAACGCAAGTCATCAGCTTGCATTGAATACG TCCCTGCCCTTTGTACACAYCGCCCGTCGCACCTACCGATTGAACGGTCCGATGAAACCATGGGATGWTT STGTTTGGATTVATTTTTSGACAKAGGCAGAACTCGGGTGAATCTTATTGTTTAGAGGAAGGTGAAGTCG TAACAAGGTTTCCGTAGTGA

Example 8. Carotenoid Characterization of the Thraustochytrid-LikeStrain G3-1

Analysis of the carotenoid content of G3-1 biomass and oil wasperformed. Carotenoid analysis, performed on biomass from three separate2 L fermentations, demonstrated that strain G3-1 synthesize and stores asmall amount of carotenoids with 0-carotene being the main components(Table 9). This contrasts with other thraustochytrids, in whichcanthaxanthin is the major carotenoid, but is similar to observationspublished for other thraustochytrid-like strains (in Lee Chang et al.,“Biodiscovery of New Australian Thraustochytrids for Production ofBiodiesel and Long-Chain Omega-3 Oils.”).

TABLE 9 Summary of carotenoid content for Thraustochytrid-like strainG3-1. Carotenoid Concentration (per Dry weight (μg g⁻¹)) Sample β- β-Day Astaxanthin Zeaxanthin Canthaxanthin Cryptoxanthin LycopeneEchinenone Carotene Replicate 1 1 0 0 0 0 0 0 20.37 7 0 0 0 0 0 0 10.379 0 0 5.14 0 0 0 9.26 Replicate 2 1 0 0 0 0 0 0 23.35 7 0 0 0 0 0 0 6.429 0 0 0 0 0 0 7.54 Replicate 3 1 0 0 0 0 0 0 10.77 7 0 0 0 0 0 0 4.68 90 0 0 0 0.13 0 7.37

Experimental Details

For the analysis of carotenoids, biomass was extracted using small glassbeads and a bead beater, in the presence of chilled acetone:methanol(1:1 v/v) twice, followed by two extractions using hexane. The combinedsupernatant was dried under nitrogen and re-suspended in acetone withantioxidants (0.5% butylated hydroxyanisole (BHA) and butylatedhydroxytolunene (BHT). Analysis was conducted on an Agilent HPLC using aPhenomenex 5μ Luna C18(2) column.

In summary, through four experiments, which include a single mediaoptimization and three 2 L fermentations, conditions were identifiedthat increased biomass productivity from 0.589 g L⁻¹ h⁻¹ to 0.740 g L⁻¹h⁻¹, and separately increase TFA synthesis from 0.396 g L⁻¹ h⁻¹ to 0.548g L⁻¹ h⁻¹. This alone exceed the best published TFA productivityby >80%, which is at 0.301 g L⁻¹ h⁻¹. What is more, within the producedoil DHA production is 0.202 g L⁻¹ hr⁻¹ and palmitic acid is 0.264 g L⁻¹h⁻¹. Our analysis suggests DHA productivity compares favorably to highlyoptimized thraustochytrid based methods, which when cultured underconditions for the production of high DHA oil, may produce around 0.3 gL⁻¹ h⁻¹.

Example 9. Identifying Key Media Ingredients for G3-1 Biomass and LipidAccumulation in Different Media Formulations Having Monosodium Glutamate(MSG) as the Defined Organic Nitrogen Source and Evaluation of the FattyAcid Profile of the G3-1 Lipids Synthesized Under these Conditions

Previous examples have showed that G3-1 requires a complex source ofnitrogen (soy peptone and yeast extract) to grow and accumulate biomass.However, soy peptone is not only an expensive source of complex organicnitrogen, the use of high concentrations of soy peptone in the media togrow some microalgae strains have led to the synthesis of odd chainsaturated fatty acids (particularly C15:0 and C17:0) in the lipidfraction of microalgae cells. For instance, G3-1 cells accumulate 15.1%of its total fatty acids as C15:0, when 20 g soy peptone L⁻¹ werepresent in the basal media that was used to grow G3-1 (Example 3). Todecrease the cost of G3-1 media and to avoid having odd chain saturatedfatty acids in the lipid fraction of G3-1 cells a Plackett-Burmanexperimental design was used to identify key ingredients that supportG3-1 cell proliferation and that must be present in a media where theuse of soy peptone has been replaced by MSG and yeast extract.

Experimental Details

A Plackett-Burman design was used to identify the significance of sixmedia ingredients on G3-1 biomass and lipid accumulation. The six mediaingredients (independent variables) were tested at a high (+) and a lowlevel (−) (Table 10). A total of twelve different media compositions forgrowing G3-1 having different combinations of ingredients were tested induplicate, as described in Table 11. Based on previous experiments andin order to favor cell proliferation and lipid synthesis, for each ofthe twelve media compositions tested, the concentration of glucose andsodium chloride (NaCl) were kept at 40 g L⁻¹ and 2, respectively.

95 mL of each media tested were made following the combination ofingredients showed in Table 11 and were inoculated with 5 mL aliquots ofa G3-1 seed flask. The composition of the media used for the seed flaskwas 20 g glucose L⁻¹, 30 g yeast extract L⁻¹ and 9 g sea salts L⁻¹.

TABLE 10 Independent variables and their levels used in thePlackett-Burman design. Coded level Variables Code Xi −1 +1 MSG (g L⁻¹)X1 0 1 Yeast extract (g L⁻¹) X2 0 1 KH2PO4 (g L⁻¹) X3 0.1 0.3 Traceelement solution X4 0 1.5 (mL⁻¹) FeCl₃(g L⁻¹) X5 0 5 MgSO₄•7H₂O(g L⁻¹)X6 0 4

TABLE 11 Plackett-Burman design matrix. Coded Variable Process variableRun Block X1 X2 X3 X4 X5 X6 X1 X2 X3 X4 X5 X6  1 1 +1 −1 +1 −1 −1 −1 1 00.3 0 0 0  2 1 +1 +1 −1 +1 −1 −1 1 1 0.1 1.5 0 0  3 1 −1 +1 +1 −1 +1 −10 1 0.3 0 5 0  4 1 +1 −1 +1 +1 −1 +1 1 0 0.3 1.5 0 4  5 1 +1 +1 −1 +1 +1−1 1 1 0.1 1.5 5 0  6 1 +1 +1 +1 −1 +1 +1 1 1 0.3 0 5 4  7 1 −1 +1 +1 +1−1 +1 0 1 0.3 1.5 0 4  8 1 −1 −1 +1 +1 +1 −1 0 0 0.3 1.5 5 0  9 1 −1 −1−1 +1 +1 +1 0 0 0.1 1.5 5 4 10 1 +1 −1 −1 −1 +1 +1 1 0 0.1 0 5 4 11 1 −1+1 −1 −1 −1 +1 0 1 0.1 0 0 4 12 1 −1 −1 −1 −1 −1 −1 0 0 0.1 0 0 0

In addition to glucose (40 g L⁻¹), sodium chloride (2 g L⁻¹), yeastextract and MSG, the most significant ingredients (p<0.05) that must beadded to the media to favor G3-1 cell proliferation were: MgSO₄.7H₂O(×6), trace elements (×4) and KH₂PO₄ (×3). The highest biomassaccumulation (13.2±0.1 g L⁻¹) was obtained using a media having at least1 g yeast extract L⁻¹, 1 g MSG L⁻¹, 0.3 g KH₂PO₄ L⁻¹, 1.5 mL L⁻¹, 4 gMgSO₄.7H₂O L⁻¹, 40 g glucose L⁻¹ and 2 g NaCl L⁻¹. FeCl₃ did not show asignificant effect (p>0.05) on G3-1 biomass accumulation, so it waseliminated from the media formulation to grow G3-1.

On the other hand, ANOVA of the lipid accumulation data for each of themedias tested by using the Plackett-Burman design showed that MgSO₄.7H₂O(×6), trace elements (×4) and KH₂PO₄ (×3) had a positive significanteffect (p<0.05) on lipid accumulation expressed as total fatty acids(TFA). MSG also had a statistically significant effect on TFAconcentration, however its effect on TFA was negative, which means thatG3-1 synthesized less lipids when the concentration of MSG in the mediawas high. This was expected, because low carbon to nitrogen ratios (C/N)affect lipid biosynthesis in G3-1 cells. The best combination of mediaingredients to obtain the highest TFA (789 mg g⁻¹) were: at least 1 gyeast extract L⁻¹, 0.3 g KH₂PO₄ L⁻¹, 1.5 mL trace elements L⁻¹, 4 gMgSO₄.7H₂O L⁻¹, 40 g glucose L⁻¹ and 2 g NaCl L⁻¹. In addition toaffecting biomass and TFA production, fatty acid profile was alsoinfluenced by the different media compositions tested by using thePlackett-Burman design (FIG. 8). For instance, media formulations forrun 10, 11 and 12 of the Plackett-Burman design matrix (Table 12) showeda very low DHA content: 1, 1.5 and 3.5, respectively; whereas C16:0content (%) was 87.5, 82.9 and 80.9, respectively. The media for runs10, 11 and 12 lack trace elements solution, this ingredient is not onlyrequired for G3-1 cell proliferation but it also has to be added to themedia to enhance lipid biosynthesis and to favor accumulation of lipidswith a more balanced fatty acid profile, such as the oily biomassobtained for run 4 and 7 (Table 12, FIG. 8).

TABLE 12 Fatty acid profile of the intracellular oil of strain G3-1under different liquid media compositions tested using a Plackett-Burmandesign. Fatty acids (%) C22:5 C22:6 n-6 n-3 Biomass TFA Run C10:0 C14:0C15:0 C16:0 C18:0 DPA DHA SFA MUFA PUFA (g/L) (g/L d)  1 3.2 3.9 3.078.0 2.7 1.6 6.0 92.3 0 7.7  0.5 ± 0.02 0.04  2 1.0 4.2 1.9 77.3 1.7 2.39.6 87.3 0.2 12.5 3.5 ± 0.2 0.5  3 1.3 5.1 8.1 73.4 1.6 1.5 5.9 92.5 07.5  1.3 ± 0.05 0.2  4 0.1 4.8 0.4 51.9 1.1 7.4 32.5 58.9 0.2 41.0 13.2± 0.1  3.5  5 0.8 3.9 1.7 73.8 1. 3.3 12.6 83.2 0.3 16.5 3.5 ± 0.1 0.4 6 1.0 6.6 1.1 85.5 2.1 0.5 2.1 97.5 0 2.5  5.8 ± 0.01 0.9  7 0.1 4.41.0 53.2 1.1 7.1 31.3 60.4 0.1 39.5 9.4 ± 0.1 2.6  8 0.4 5.2 2.4 76.81.5 2.5 9.3 87.6 0.1 12.2 3.3 ± 0.1 0.6  9 0.2 5.6 0.6 67.2 1.3 3.5 18.975.5 0.1 24.3 6.2 ± 0.2 1.4 10 1.2 6.2 1.0 87.5 1.9 0.3 1.0 98.6 0.2 1.35.9 ± 0.2 0.9 11 3.1 6.1 3.3 82.9 2.0 ND 1.5 98.5 0 1.5 3.1 ± 0.2 0.5 121.6 5.9 4.2 80.9 1.6 0.9 3.5 95.6 0 4.4  2.5 ± 0.04 0.4 ND notdetectable

Example 10. The Effect of a Complex Nitrogen Source (Yeast Extract), aSimple Organic Nitrogen Source (MSG) and a Simple Inorganic NitrogenSource (Ammonium Sulfate, (NH₄)₂SO₄) on G3-1 Biomass Accumulation

G3-1 cells showed the ability to accumulate biomass in a liquid medialacking soy peptone but having MSG and yeast extract as the simple andcomplex organic nitrogen sources, respectively. Previous examplesdescribed in this document have demonstrated that G3-1 requires highconcentrations of complex and simple organic nitrogen in order toaccumulate a decent amount of biomass. Formulation of media anddevelopment of fermentation strategies to produce high concentrations ofoily G3-1 biomass are imperative when developing microalgae fermentationtechnologies to produce value-added products, such as DHA, protein,carotenoids, etc.

Experimental Details

In this example, the effect of three different nitrogen sources (yeastextract, MSG and (NH₄)₂SO₄) on biomass accumulation by G3-1 were testedby using a two-level full factorial design (2³). The concentration ofthe three nitrogen sources (independent variables) were tested at a high(+) and a low level (−) (Table 13). A total of eight different mediacompositions for growing G3-1 having different combinations of yeastextract, MSG and (NH₄)₂SO₄ were tested in duplicate, as described inTable 14. Based on previous examples and in order to favor cellproliferation, for each of the eight media compositions tested, theconcentration of glucose, NaCl, MgSO₄.7H₂O, trace elements solution,KH₂PO₄, K₂HPO₄, CaCl₂) and vitamin B solution were kept at 40 g L⁻¹, 2 gL⁻¹, 4 g L⁻¹, 1.5 mL L⁻¹, 1.6 g L⁻¹, 1.74 g L⁻¹, 0.5 mL L⁻¹ and 1 mLL⁻¹, respectively. 95 mL of each media tested were made following thecombination of ingredients showed in Table 14 and were inoculated with 5mL aliquots of pure washed G3-1 cultures.

TABLE 13 Independent variables and their levels used in the fullfactorial design (2³). Coded level Variables Code Xi −1 +1 Yeast extract(g L⁻¹) X1 5 15 MSG (g L⁻¹) X2 1 20 (NH₄)₂SO₄(g L⁻¹) X3 5 10

TABLE 14 Full factorial 2³ design matrix and biomass results. CodedVariable Process variable Biomass Run Block X1 X2 X3 X1 X2 X3 (g/L) 1 1−1 −1 −1 5 1 5 11.5 ± 0.2  2 1 +1 −1 −1 15 1 5 18.5 ± 0.2  3 1 −1 +1 −15 20 5 29.5 ± 0.6  4 1 +1 +1 −1 15 20 5 32.5 ± 0.2  5 1 −1 −1 +1 5 1 1010.5 ± 1.0  6 1 +1 −1 +1 15 1 10 19.0 ± 0.3  7 1 −1 +1 +1 5 20 10 29.8 ±0.3  8 1 +1 +1 +1 15 20 10 30.7 ± 0.4 

The ANOVA of the biomass data showed that the most statisticallysignificant (p<0.05) nitrogen sources influencing biomass accumulationwere: MSG and yeast extract. The interaction between MSG and yeastextract was also significant (p<0.05), which means that, G3-1 cellsprefers to uptake MSG first and then start to use yeast extract as thenitrogen source. The highest biomass concentration for this example(32.5±0.5 g L⁻¹) was obtained when G3-1 was grown in a media having 15 gyeast extract L⁻¹, 20 g MSG L⁻¹ and 5 g (NH₄)₂SO₄ L⁻¹ (run 4 Table 14).Run 8 also showed a good accumulation of biomass (30.7±0.4 g L⁻¹),however this media formulation requires 50% more (NH₄)₂SO₄ compared torun 4.

Based on the results described on this example, media formulations forrun 4 and 8 were named VU1 and VU2, respectively, and were selected todevelop fermentation processes to obtain two different G3-1 biomassproducts: (1) oily biomass having at least 60% lipids and 35% C22:6n-3(DHA) in the triglycerides in the total fatty acids, and (2) biomasshaving at least 18% true protein in the whole algae biomass. Trueprotein is expressed as the sum of amino acid concentrations in thebiomass sample.

Example 11. Production of Omega-3 Rich Oily Biomass

VU1 media formulation was selected to carry out a fermentation using a30 L fermenter to obtain omega-3 rich oily biomass with applications inaquafeed. In this 116.5 h fermentation G3-1 produced 98.4 g L⁻¹ biomasscomposed of 66.2% TFA and 8% true protein. DHA and palmitic acidconstituted 39.4% and 46.1% of TFA, respectively.

Experimental Details

G3-1 was pre-cultured in Erlenmeyer flasks containing 500 mL of seedflask media (20 g glucose L⁻¹, 5 g yeast extract L⁻¹, 2 g NaCl L⁻¹, 4 gMgSO₄ L⁻¹, 3 mg copper sulfate L⁻¹, 2 mg sodium molybdate L⁻¹, 3 mg zincsulfate L⁻¹, 2 mg cobalt (II) chloride L⁻¹, 2 mg manganese chloride L⁻¹and 2 mg nickel sulfate L⁻¹). Flasks were incubated under agitation at25° C. and 200 rpm for 2 days. After the incubation period, 1 L of thepre-cultured cells were transferred into a 19 L of VU1 media. Thecomposition of VU1 media per liter was: 15 g yeast extract, 20 g MSG, 5g (NH₄)₂SO₄, 40 g glucose, 2 g NaCl, 4 g MgSO₄, 1.6 g KH₂PO₄, 1.75 gK₂HPO₄, 3 mg copper sulfate, 2 mg sodium molybdate, 3 mg zinc sulfate, 2mg cobalt (II) chloride, 2 mg manganese chloride, 2 mg nickel sulfate,0.1 g calcium chloride dehydrate, 0.01 g cobalamin, 0.01 g biotin and 2g thiamin hydrochloride. A batch fermentation was carried out in a 30 Lfermenter under the following conditions: 25° C., pH 6.8, aeration at0.5 VVM with atmospheric air, agitation starting at 357 rpm and reaching447 rpm. Cells were collected at 10-18 h intervals and the biomass, TFA,DHA and protein were measured. The initial glucose in the media wascompletely depleted after 24 h of fermentation and at that time theculture was then fed with 75% (w/v) glucose, until 116.5 h when thefermentation was ended. For this fermentation productivity for biomass,TFA and DHA was: 0.84 g L⁻¹ h⁻¹, 0.56 g L⁻¹ h⁻¹ and 0.22 g L⁻¹ h⁻¹,respectively (Table 15, FIG. 9). On the other hand, biomass at the endof the fermentation had 8% true protein.

TABLE 15 Oil production and fatty acid profile of the intracellular oilof MARA G3-1. Cells were cultured in a 30 L fermenter using VU1 media.Fatty acids (%) C20:5 C22:5 C22:6 Time TFA (n-3) (n-6) (n-3) Biomass TFAProductivity (g/L h) (h) (%) C14:0 C15:0 C16:0 C18:0 EPA DPA DHA (g/L)(mg/g) Biomass TFA DHA C16:0 22 27 2.7 0.6 43.4 1.3 0.3 6.7 42 26 269.548 44.1 3.3 0.4 44.5 1.3 0.4 6.7 40.4 57 441.3 72.5 57.7 2.8 0.2 42.41.3 0.4 7.3 42.5 87.4 577.3 96 60.6 3.2 0.2 46.7 1.4 0.4 6.7 38.6 96.8605.9 116.5 66.2 3.1 0.2 46.1 1.4 0.5 6.7 39.4 98.4 662.2 0.84 0.56 0.220.26

The fermentation technology developed to produce G3-1 omega-3 rich oilybiomass can easily achieve ≥15 g/L d productivity in biomass, 66% lipidand >39% DHA in 4.9 days. A biomass with this composition could be usedto feed fish in in vivo trials to assess its suitability as a DHA richaquafeed product for farmed fish.

Example 12. Production of Omega-3 Rich Microalgae Biomass with a HigherProtein Content

VU2 media formulation was selected to carry out a fermentation using a30 L fermenter to obtain omega-3 rich biomass high in protein. A biomassproduct having high DHA and high protein while still containing 30-45%lipids has the potential to be used as an aquafeed product. In this 72 hfermentation G3-1 produced 93.4 g L⁻¹ biomass composed of 43.6% TFA,47.8% DHA, 36.9% palmitic acid and 18.6% true protein.

Experimental Details

G3-1 was pre-cultured in Erlenmeyer flasks containing 500 mL of seedflask media (20 g glucose L⁻¹, 5 g yeast extract L⁻¹, 2 g NaCl L⁻¹, 4 gMgSO₄ L⁻¹, 3 mg copper sulfate L⁻¹, 2 mg sodium molybdate L⁻¹, 3 mg zincsulfate L⁻¹, 2 mg cobalt (II) chloride L⁻¹, 2 mg manganese chloride L⁻¹and 2 mg nickel sulfate L⁻¹). Flasks were incubated under agitation at25° C. and 200 rpm for 2 days. After the incubation period, 1 L of thepre-cultured cells were transferred into a 19 L of VU2 media. Thecomposition of VU2 media per liter was: 15 g yeast extract, 20 g MSG, 10g (NH₄)₂SO₄, 40 g glucose, 2 g NaCl, 4 g MgSO₄, 1.6 g KH₂PO₄, 1.75 gK₂HPO₄, 3 mg copper sulfate, 2 mg sodium molybdate, 3 mg zinc sulfate, 2mg cobalt (II) chloride, 2 mg manganese chloride, 2 mg nickel sulfate,0.1 g calcium chloride dehydrate, 0.01 g cobalamin, 0.01 g biotin and 2g thiamin hydrochloride. A batch fermentation was carried out in a 30 Lfermenter under the following conditions: 25° C., aeration at 0.5 VVMwith atmospheric air, agitation starting at 357 rpm and reaching 370rpm. Finally, the pH of the culture was kept around 6.2 by using anaqueous solution of ammonium hydroxide. Cells were collected at 6-18 hintervals and the biomass, TFA, DHA and protein were measured. Theinitial glucose in the media was completely depleted after 22 h offermentation and at that time the culture was then fed with 75% (w/v)glucose, until 72 h when the fermentation was ended. For thisfermentation productivity for biomass, TFA and DHA was: 1.3 g L⁻¹ h⁻¹,0.57 g L⁻¹ h⁻¹ and 0.27 g L⁻¹ h⁻¹, respectively (Table 16, FIG. 10).

TABLE 16 Oil production and fatty acid profile of the intracellular oilof MARA G3-1. Cells were cultured in a 30 L fermenter using VU2 media.Fatty acids (%) C20:5 C22:5 C22:6 True Time TFA (n-3) (n-6) (n-3)Biomass TFA Productivity (g/L h) protein (h) (%) C14:0 C15:0 C16:0 C18:0EPA DPA DHA (g/L) (mg/g) Biomass TFA DHA C16:0 (%) 24 17 1.9 0.4 37.11.2 0.3 8.2 47.4 27.8 170.4 42 25.8 2.5 0.3 39.6 1.2 0.4 7.7 44.9 54.2257.8 48 30.2 2.5 0.2 37.7 1.1 0.4 8.0 46.9 62.4 302.2 66 42.7 3.0 0.237.3 1.0 0.4 7.5 47.5 86.0 426.6 72 43.6 2.9 0.2 36.9 1.0 0.4 7.6 47.893.4 435.6 1.3 0.57 0.27 0.21 18.6

The fermentation process described in this example produced a G3-1biomass product not only rich in omega-3 DHA but also with a higherprotein content compared to the product described as omega-3 rich oilyG3-1 biomass. Growing G3-1 in VU2 media and using an aqueous solution ofammonium hydroxide to control the pH and also to pulse nitrogen into thevessel not only modified the C/N ratio in the fermentation broth butalso, favored nitrogen metabolic pathways in the cells which in turnlimited the ability of G3-1 to synthesized lipids, improving G3-1protein content. 31.1 g/L d productivity in biomass, 43.6% lipid, >47%DHA and 18.6% true protein can easily be achieved in 3 days by followingthe fermentation technology described in this example.

Example 13. Production of G3-1 Biomass Low in Lipids and Enhancement ofDHA and EPA (Expressed as Percentage of Total Fatty Acids) by UsingCrude Glycerol from Biodiesel Production

The potential use of crude glycerol to cultivate oleaginousmicroorganisms has grown in popularity, essentially to reducecultivation costs. Simultaneously, the need to valorize glycerol as aco-product of biodiesel has arisen as a result of the biofuel boom.Depending on the feedstock and the process used to produce biodiesel,the contaminants present in crude glycerol vary. The most common onesare methanol and soap, but also high salinity. As a consequence, tons ofraw glycerol need to be valorized or classified as industrial waste. Thehigh salinity of crude glycerol has undesired effects on many organism,however, it favors the use of marine microalgae. Fermentation processesbased on crude glycerol as the carbon source aiming to obtain higherquantities of value-added metabolites, such as PUFAs, particularly EPAand DHA are the ones with the highest value (Abad and Turon, Mar. Drugs.13:7275 (2015)). Previous efforts to use crude glycerol in marinemicroalgae fermentation bioprocesses have focused on the production ofhigh concentrations of intracellular lipids, however, there is littleinformation regarding the use of a fermentation process to delay oilaccumulation in oleaginous microorganisms while using crude glycerol asthe carbon source. In this example, a crude glycerol feedstock having1120 g glycerol L⁻¹ was used in combination with a liquid mediaformulation called VU3 to grow G3-1 under heterotrophic conditions. Inthis 118.5 h fermentation G3-1 produced 61.7 g L⁻¹ biomass composed of15.6% TFA, 55% DHA, 19.7% palmitic acid and 4.1% EPA.

Experimental Details

G3-1 was pre-cultured in Erlenmeyer flasks containing 250 mL of seedflask media (20 g glucose L⁻¹, 5 g yeast extract L⁻¹, 2 g NaCl L⁻¹, 4 gMgSO₄ L⁻¹, 1 g MSG L⁻¹, and 3 mg copper sulfate L⁻¹, 2 mg sodiummolybdate L⁻¹, 3 mg zinc sulfate L⁻¹, 2 mg cobalt (II) chloride L⁻¹, 2mg manganese chloride L⁻¹ and 2 mg nickel sulfate L⁻¹). Flasks wereincubated under agitation at 25° C. and 200 rpm for 2 days. After theincubation period, 0.14 L of the pre-cultured cells were transferredinto 1.26 L of VU3 media. The composition of VU3 media per liter was: 2g yeast extract, 5 g MSG, 10 g (NH₄)₂SO₄, 66 g crude glycerol, 2 g NaCl,4 g MgSO₄, 1.6 g KH₂PO₄, 1.75 g K₂HPO₄, 3 mg copper sulfate, 2 mg sodiummolybdate, 3 mg zinc sulfate, 2 mg cobalt (II) chloride, 2 mg manganesechloride, 2 mg nickel sulfate, 0.1 g calcium chloride dehydrate, 0.01 gcobalamin, 0.01 g biotin and 2 g thiamin hydrochloride. A batchfermentation was carried out in a 2 L fermenter under the followingconditions: 25° C., aeration at 1 VVM with atmospheric air, agitationstarting at 550 rpm and reaching 710 rpm. During the first 65.75 h offermentation the pH of the culture was kept around 6.16-6.26 by using anaqueous solution of ammonium hydroxide. Then the ammonium hydroxidesolution was swapped by 5 M NaOH to keep the pH around 6.05 up to 188.5h of fermentation. Cells were collected at 6-18 h intervals and thebiomass, TFA, palmitic acid, DHA and EPA were measured. The initialglucose in the media was completely depleted after 40 h of fermentationand at that time the culture was then fed with 1120 g crude glycerolL⁻¹, until 188.5 h when the fermentation was stopped. At the end of theprocess productivity for biomass and TFA was 0.33 g L⁻¹ h⁻¹ and 0.05 gL⁻¹ h⁻¹. On the other hand, DHA and EPA content (expressed as % of TFA)was 55% and 4.1%. In previous examples G3-1 has shown a poor ability toaccumulate EPA in its lipid fraction with contents ranging from 0.5 to1% of TFA when glucose was used as the carbon source, however under theconditions described in this example, the amount of EPA synthesized byG3-1 was 3 times higher (Table 17, FIG. 11). Adverse culture conditionssuch as nutrient deficiency or toxic compounds in the liquid mediastimulated the synthesis of EPA by thraustochytrids. It is hypothesizedthat the biosynthesis of EPA and other PUFAs by thraustochytrids is toprovide antioxidant power to protect the cells when subjected tooxidative stress (Ugalde et al., J. Appl. Phycol. (2017)).

TABLE 17 Oil production and fatty acid profile of the intracellular oilof MARA G3-1. Cells were cultured in a 2 L fermenter using VU3 media andcrude glycerol as the carbon source. Fatty acids (%) C20:5 C22:5 C22:5C22:6 Time TFA C18:1 (n-3) (n-6) (n-3) (n-3) Biomass TFA Productivity(g/L h) (h) (%) C14:0 C16:0 C18:0 oleic EPA DPA DPA DHA (g/L) (mg/g)Biomass TFA DHA C16:0 40.5 17.9 2.0 30.8 1.1 0.2 0.8 9.5 0.3 51.7 36.9178.7 65.75 12.4 1.0 20.8 0.7 0.2 2.8 11.6 0.8 58.8 27.8 124.2 92.5013.3 1.6 24.1 0.8 0.7 3.5 10.9 1.0 53.0 47.6 132.7 113.5 14.4 1.4 22.60.8 1.1 3.8 11.0 1.1 53.2 53.9 143.5 137 15.1 1.3 21.7 0.8 1.2 4.0 11.11.2 53.6 57.4 151.0 163.75 15.1 1.2 20.3 0.8 1.2 4.1 11.4 1.3 54.6 59.8151.3 188.5 15.6 1.2 19.7 0.8 1.2 4.1 11.5 1.4 55.0 61.7 155.9 0.33 0.050.03 0.01

74.5 mL of an ammonium hydroxide solution were pulsed into the 2 Lvessel during the first 65.75 h of fermentation. From 65.75 h to 188.5 hthe ammonium hydroxide solution was swapped for a solution of NaOH 5 Mto keep pH around 6.05 and push G3-1 cells to accumulate lipids. As seenon Table 17 pulsing nitrogen in the form of ammonium hydroxide helped toincrease the biomass content, however, swapping ammonium hydroxidesolution for NaOH 5M and keeping the fermentation running for an extrafive days did not favor lipid accumulation in G3-1 cells. G3-1 cellsuptake nitrogen in the form of ammonium hydroxide but it seems thatcatabolism of ammonium hydroxide interferes with the lipid biosynthesispathways in G3-1. On the other hand, not only ammonium sulfate but thecrude glycerol used as a carbon feedstock could have stressed G3-1cells. The crude glycerol used in the present example, contains 0.8%methanol and a high salinity, these impurities act as stress factorseven for marine microalgae that can tolerate high salinity environments.It has been reported that abiotic stress often causes amino acids, whichserve as potential stress mitigators, to accumulate. In addition tobeing the building blocks of proteins, amino acids serve as theprecursors of N-containing molecules such as nucleic acids, polyamines,quaternary ammonium compounds, and some hormones. Under environmentalstress, de novo protein synthesis is generally inhibited and proteinturnover and proteolytic activity are increased, resulting in anincrease of total free amino acids. N and C metabolisms are closelyconnected; N assimilation and amino acid biosynthesis require reducingequivalents from carbon metabolism (e.g., glucose, glycerol, etc.) and Cskeletons from the tricarboxylic acid (TCA) cycle (Chen et al.,Biotechnol. Biofuels 10:153 (2017)). At the end of the fermentation(e.g., at 188.5 h) G3-1 cells accumulated 15.6% of intracellular lipids(Table 17) even though G3-1 cells consumed 160.5 g of crude glycerol. Itseems that the energy and C skeletons generated from the crude glycerolmetabolism were not used for lipid accumulation but were redirected todifferent metabolic pathways in G3-1 cells.

1.-27. (canceled)
 28. A method of making a lipid composition, the methodcomprising (a) culturing a eukaryotic microorganism in a heterotrophicmedium to produce a biomass comprising the lipid composition comprisingat least 35% DHA and a simple lipid profile comprising long chain fattyacids (LCFAs), wherein the simple lipid profile comprises triglyceridesand wherein greater than 95% of the triglycerides are comprised ofmyristic acid (C14:0), palmitic acid (C16:0), docosapentaenoic acid n-6(C22:5n-6, DPAn6), and docosahexaenoic acid (C22:6n-3, DHA), (b) andisolating the lipid composition.
 29. The method of claim 28, wherein theheterotrophic medium contains less than 3.75 g/L chloride.
 30. Themethod of claim 28, wherein the biomass productivity of the culturedmicororganisms is greater than 0.65 g/L/h.
 31. The method of claim 28,wherein the triglyceride productivity of the cultured microorganisms isgreater than 0.3 g/L/h. 32.-36. (canceled)
 37. The method of claim 28,wherein the simple lipid profile comprises less than 3% of each oflauric acid (C12:0), pentadecylic acid (C15:0), palmitoleic acid(C16:1), margaric acid (C17:0), stearic acid (C18:0), vaccenic acid(C18:1n-7), oleic acid (C18:1n-9), γ-linolenic acid (C18:3n-6),α-linolenic acid (C18:3n-3), stearidonic acid (C18:4), arachidic acid(C20:0), dihomo-γ-linolenic acid (C20:3n-6), arachidonic acid (C20:4n-6,ARA), eicosapentaenoic acid (C20:5n-3, EPA), behenic acid (C22:0),docosatetraenoic acid (C22:4), docosapentaenoic acid n3 (C22:5n-3,DPAn3), and lignoceric acid (C24:0).
 38. The method of claim 28, whereinthe simple lipid profile comprises less than 0.02% short chain fattyacids.
 39. The method of claim 28, wherein the biomass comprises atleast 20% protein.
 40. The method of claim 28, wherein the biomasscomprises at least 20 to 40% protein.
 41. The method of claim 28,wherein the biomass comprises at least about 40% protein.
 42. The methodof claim 28, wherein the composition comprises at least 30% palmiticacid.
 43. The method of claim 28, wherein the composition comprises atleast 40% palmitic acid.
 44. The method of claim 28, wherein the biomasscomprises one or more carotenoids.
 45. The method of claim 44, whereinthe one or more carotenoids comprises 3-carotene, and wherein the0-carotene comprises at least 95% of total carotenoids.
 46. The methodof claim 28, further comprising incorporating the lipid composition intoa foodstuff.
 47. The method of claim 46, wherein the foodstuff is petfood, a livestock feed, or an aquaculture feed.
 48. The method of claim28, further comprising incorporating the lipid composition into anutraceutical or pharmaceutical product.
 49. The method of claim 28,further comprising incorporating the lipid composition into a dietarysupplement.
 50. The method of claim 28, wherein the lipid compositioncomprises 35% to 50% DHA.
 51. The method of claim 28, wherein the lipidcomposition comprises 40% to 50% DHA.
 52. The method of claim 28,wherein the lipid composition comprises 50% to 60% DHA.